Small molecules that mimic or antagonize actions of granulocyte colony-stimulating-factor (g-csf)

ABSTRACT

Described herein are compound for the modulation of the G-CSF receptor. The compounds may act as agonists, antagonists, and/or mixed or partial agonists/antagonists of G-CSF. Further provided herein are methods of treating a condition, including, for example, a neurodegenerative disease, by administering a compound as detailed herein.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/426,032, filed Nov. 23, 2016, which is incorporated herein byreference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 11,580 Byte ASCII (Text) file named“16B135-210112-9006-US01-SEQ-LIST-11-22-17.txt” created on Nov. 22,2017.

FIELD

This disclosure relates to Granulocyte Colony-Stimulating Factor (G-CSF)and treatments for neurodegenerative disease.

INTRODUCTION

Granulocyte colony-stimulating factor (G-CSF) is a hematopoieticcytokine commonly used for treatment of neutropenia and to increasegeneration of hematopoietic stem/progenitor cells in bone marrow donors.G-CSF also exerts direct effects on neural stem/progenitor cells and isexpressed, along with its receptor G-CSF-R, in neurogenic zones of thehippocampus (HC), the sub-ventricular zone, and the olfactory bulb. TheG-CSF receptor and its ligand are also expressed by mature neurons inseveral other areas of the brain including pyramidal cells in corticallayers (specifically II and V), entorhinal cortex, Purkinje cells of thecerebellum, and in cerebellar nuclei in rats. The G-CSF receptor iscommon and can be found on the cell surfaces of endothelial cells,lymphocytes, platelets, and neutrophils.

The role of G-CSF as a neurotrophic factor during development and inadult life is increasingly recognized. Mice bred to have a deficiency inG-CSF were reported to have problems with memory formation anddevelopment of motor skills. More specifically, the hippocampus fromthese mice exhibited deficits in the induction of long term potentiationin the CA1 region, decreased neuronal precursor cells in the dentategyrus (DG), and decreased dendritic complexity in neurons in the DG andCA1 region of the HC. The defects seen in G-CSF deficient mice supportthe designation of G-CSF as a true neurotrophic factor, playing a rolein neurogenesis and the maintenance of structural and functionalintegrity of the hippocampal formation. In combination with cognitivetraining, G-CSF can also significantly improve spatial learning and newneuron survival in the hippocampus.

The recognition that G-CSF acts directly on neural tissue has stimulatedresearch on the therapeutic applications of these agents forneurodegenerative diseases, stroke, and brain trauma. G-CSFadministration was reported to decrease amyloid burden, enhanceneurogenesis, synaptogenesis, and cognitive performance in a mouse modelof Alzheimer's disease (AD). Systemic G-CSF administration to rats thathad sustained traumatic brain injury (TBI) resulted in significantlybetter motor function recovery than the control group. G-CSFadministration to mice in a model of traumatic brain injury resulted inenhanced recovery of cognitive function in a hippocampal-dependentlearning task. G-CSF also improves survival in a transgenic mouse modelof amyotrophic lateral sclerosis (ALS). In a mouse model of AD (thetransgenic APP/PS1 mouse), G-CSF treatment was effective in decreasingamyloid burden, enhancing neurogenesis, and improving performance on ahippocampal-dependent task.

Granulocyte-colony stimulating factor (G-CSF) is both a hematopoieticgrowth factor used clinically to treat neutropenia and a neurotrophicfactor that exerts direct effects on neural cells. There are severaldisadvantages to the application of human G-CSF as a neurotrophic factorto treat neurologic diseases. For example, (1) human G-CSF is producedby recombinant DNA technology and may be expensive to manufacture andcostly to administer as a course of treatment; (2) the primaryperipheral actions of G-CSF limit the dose that can be safelyadministered to treat brain disorders; and (3) there are presently noknown specific G-CSF receptor antagonists capable of blocking theperipheral actions of G-CSF, leaving intact the direct neurotrophiceffects in brain. Small molecule mimetics of G-CSF are highly sought aspotential drugs for neurodegenerative diseases, stroke, and brain injurytrauma.

SUMMARY

In an aspect, the disclosure relates to methods of treating a conditionin a subject. The method may include administering a compound to thesubject, wherein the compound is according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula II:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy, and wherein thecondition is selected from the group consisting of neurodegenerativedisease, stroke, traumatic brain injury (TBI), impaired motor function,and impaired cognitive function.

In some embodiments, the neurodegenerative disease is selected from thegroup consisting of Alzheimer's Disease (AD), amyotrophic lateralsclerosis (ALS), Parkinson's Disease (PD), prion disease, motor neurondisease, Huntington's Disease, spinocerebellar ataxia, and spinalmuscular atrophy.

In a further aspect, the disclosure relates to methods of stimulatingthe central nervous system Granulocyte Colony-Stimulating Factor (G-CSF)Receptor in a subject. The method may include administering a compoundto the subject, wherein the compound is according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula II:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy.

In some embodiments, the compound is of Formula I, and wherein R¹ and R²are each hydrogen. In some embodiments, the compound is of Formula I,and wherein R¹ and R² together with the carbon atoms to which they areattached form a phenyl ring. In some embodiments, the compound is ofFormula I, and wherein R³ and R⁴ are each hydrogen. In some embodiments,the compound is of Formula I, and wherein R³ and R⁴ together with theatoms to which they are attached form a six-membered heterocyclic ring,wherein the ring is unsubstituted or substituted with one substituentselected from amino, nitro, methyl, ethyl, and hydroxyl. In someembodiments, the six-membered heterocyclic ring is substituted withamino or nitro. In some embodiments, the compound is of Formula I, andwherein X is —C(R⁵)(R⁶)-phenyl wherein the phenyl is substituted with R⁷and R⁸. In some embodiments, the compound is of Formula I or FormulaIII, and wherein R⁵ and R⁶ are each independently hydroxyl or methyl. Insome embodiments, the compound is of Formula I or Formula III, andwherein R⁵ and R⁶ together form an oxo group. In some embodiments, thecompound is of Formula I or Formula III, and wherein R⁷ is hydroxyl orC₁-C₁₂ alkoxy. In some embodiments, R⁷ is hydroxyl. In some embodiments,the compound is of Formula I or Formula III, and wherein R⁸ is halogen.In some embodiments, halogen is Cl. In some embodiments, the compound isof Formula I or Formula II, and wherein X is indole substituted with 0,1, 2, or 3 R⁹. In some embodiments, each R⁹ is independently halogen ormethoxy. In some embodiments, the compound is of Formula I or FormulaII, and wherein X is pyrazole substituted with phenyl, wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰. In some embodiments, eachR¹⁰ is independently hydroxyl, methoxy, or nitro.

In some embodiments, the compound decreases amyloid burden, enhancesneurogenesis, enhances synaptogenesis, or enhances cognitiveperformance, or a combination thereof. In some embodiments, the compoundbinds the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor. Insome embodiments, the compound displaces at least 50% of G-CSF from theG-CSF receptor. In some embodiments, the compound displaces at least 75%of G-CSF from the G-CSF receptor. In some embodiments, the compound is aperipheral antagonist of the G-CSF receptor. In some embodiments, thecompound is a central agonist of G-CSF receptor. In some embodiments,expression of Bcl2 is increased. In some embodiments, expression ofPKCδVIII is increased. In some embodiments, expression of STAT3 isincreased. In some embodiments, expression of Bax is decreased. In someembodiments, leukopoiesis is minimally affected or is not affected.

In some embodiments, the compound is selected from the following:

In some embodiments, the compound is selected from the following:

In some embodiments, the compound is selected from the followingcompounds:

In some embodiments, the compound is co-administered with a G-CSFpolypeptide. In some embodiments, the G-CSF polypeptide comprises anamino acid sequence of SEQ ID NO: 1. In some embodiments, the G-CSFpolypeptide is encoded by a polynucleotide of SEQ ID NO: 2.

In a further aspect, the disclosure relates to a compound selected fromthe following:

The disclosure provides for other aspects and embodiments that will beapparent in light of the following detailed description and accompanyingFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical structures of ten small molecules with potential tointeract with the G-CSF receptor.

FIG. 2. Compounds 6 and 10 displaced radio-labeled G-CSF from receptorexpressed on human monocytes. Results are based on the mean of twoseparate experiments with replicates of three within each experiment.Panel on the left shows percent of radio-labeled G-CSF bound to receptorin the presence of increasing concentrations of Compound 6 and Compound10. Right panel shows Logit Y (logistic regression analysis) vsconcentrations of Compounds 6 and 10. Insert shows parameters ofcompetitive binding for Compound 6 (13.7 nM) and Compound 10 (2.3 nM).

FIG. 3A and FIG. 3B. Displacement of [I¹²⁵]-G-CSF from its receptorexpressed on monocytes by Compounds 1-10. The Y-axis in FIG. 3A showsamount of bound G-CSF in CPM and the Y-axis in FIG. 3B indicates percentof the radiolabeled G-CSF bound to receptor. (*) Indicates significantdisplacement p<0.05. Compound 10 was the most effective, displacing86.6% of I¹²⁵-G-CSF from its receptor.

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D. Western blot of PKCδVIII andBcl2 expression in (FIG. 4A) monocytic cell lines (THP-1) and (FIG. 4B)human neuronal (SH-SY5Y) incubated with G-CSF for 24 h (100 ng/mL or 200ng/mL). Both PKCδVIII and Bcl2 expression are increased in the neuronalcells (100 ng/mL; 5.3 nM), and in the monocytic cells (200 ng/mL; 10.6nM). The above graphs (FIG. 4C) and (FIG. 4D) show PKCδVIII or Bcl2densitometric units normalized to GAPDH, (Bcl1 or PKCδVIII densitometricunits÷GADPH densitometric units x 100) and represent three separateexperiments. The results were analyzed with two-tailed Student's t-test;(*) indicates p<0.0001 comparing G-CSF treatment to correspondinguntreated controls.

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D. Western blots of PKCδVIII andBcl2 expressed in SH-SY5Y neuronal cells. (FIG. 5A) Immunoblot fromcells incubated with three concentrations of Compound 6 alone and in thepresence of G-CSF (100 ng/mL). (FIG. 5B) Immunoblot from cells incubatedwith G-CSF alone and 3 concentrations of Compound 10 alone and in thepresence of G-CSF (100 ng/mL). (FIG. 5C, FIG. 5D) Densitometric analysesof Immunoblots from FIG. 5A (Compound 6) and FIG. 5B (Compound 10)respectively. (*)=p<0.05 based on one-way ANOVA of the Bcl2 datafollowed by Dunnett's multiple comparisons against vehicle control.(#)=p<0.05 based on one-way ANOVA of the PKCδVIII data alone followed byDunnett's multiple comparisons against vehicle control. Insert box showseffects of co-administration of G-CSF with Compounds 6 or 10. ($)=p<0.05based on one-way ANOVA followed by Dunnett's multiple comparisons toG-CSF alone.

FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D. Western blots of PKCδVIII andBcl2 expressed in THP-1 monocytic cells. (FIG. 6A) Immunoblot from cellsincubated with three concentrations of Compound 6 alone and in thepresence of G-CSF (100 ng/mL). (FIG. 6B) Immunoblot from cells incubatedwith 3 concentrations of Compound 10 alone and in the presence of G-CSF(100 ng/mL). (FIG. 6C, FIG. 6D) Densitometric analyses of Immunoblotsfrom FIG. 6A (Compound 6) and FIG. 6B (Compound 10), respectively.(*)=p<0.05 based on one-way ANOVA of the Bcl2 data followed by Dunnett'scorrection for multiple comparisons against vehicle control. (#)=p<0.05)based on one-way ANOVA of the PKCδVIII data alone followed by Dunnett'scorrection for multiple comparisons against vehicle control. Insert boxshows effects of co-administration of G-CSF with Compounds 6 or 10.(&)=p<0.05 based on one-way ANOVA of Bcl2 data followed by Dunnett'scorrection for multiple comparisons to G-CSF alone. ($)=p<0.05 based onone-way ANOVA of the PKCδVIII data alone followed by Dunnett'scorrection for multiple comparisons against G-CSF treatment.

FIG. 7. Schematic of PKCδ domains and alternative splicing of pre-mRNAwhich generates the splice variants PKCδI and PKCδII in mice.

DETAILED DESCRIPTION

Described herein are compounds and their use in treating variousdisorders including neurodegenerative disorders. Using computer-assisted3D molecular modeling and the crystal structure of granulocyte-colonystimulating factor receptor (G-CSF-R), small molecules were fit into thebinding site(s) of the natural ligand for the G-CSF-R. A site andmechanism by which the protein/protein interaction can be blocked withsmall molecules was discovered. Several small molecule compounds wereidentified. These small molecules were used to investigate bindingcharacteristics and the capacity to trigger intracellular signalssimilar to those activated by G-CSF. The compounds as disclosed hereinmay be agonists, antagonists, or partial agonists/antagonists of theG-CSF-R. The compounds as disclosed herein may be used, either alone orin combination with G-CSF, in methods of treating various disordersincluding neurodegenerative disorders. Effects elicited may includeinteracting or binding with the G-CSF receptor, triggering signaltransduction, acting as a neurotrophic factor, stimulating neurons thatbear G-CSF receptor, and blocking the peripheral effects of G-CSF toprevent excessive leukocytosis. The compounds described herein provideadvantages over methods of administering G-CSF directly, as G-CSF isexpensive to manufacture and costly to administer, the primaryperipheral actions of G-CSF (such as to stimulate hematopoiesis andincrease circulating levels of polymorphonucleocytes) limit the dosesthat can be used safely to treat brain disorders, and there arecurrently no known specific G-CSF receptor antagonists capable ofblocking the peripheral actions of G-CSF, leaving intact the directneurotrophic effects in the brain.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

The term “about” as used herein as applied to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain aspects, the term “about” refers to a range of values thatfall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

The term “alkoxy” or “alkoxyl” as used herein, refers to an alkyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. The term “C₁-C₁₂ alkoxy” means a C₁-C₁₂ alkyl groupappended to the parent molecular moiety through an oxygen atom. The term“C₁-C₄ alkoxy” means a C₁-C₄ alkyl group appended to the parentmolecular moiety through an oxygen atom. Representative examples ofalkoxy include, but are not limited to, methoxy, ethoxy, propoxy,2-propoxy, butoxy, and tert-butoxy.

The term “alkyl” as used herein, means a straight or branched, saturatedhydrocarbon chain containing from 1 to 20 carbon atoms. The term “C₁-C₁₂alkyl” means a straight or branched chain hydrocarbon containing from 1to 12 carbon atoms. The term “lower alkyl” or “C₁-C₄ alkyl” means astraight or branched chain hydrocarbon containing from 1 to 4 carbonatoms. The term “C₁-C₃ alkyl” means a straight or branched chainhydrocarbon containing from 1 to 3 carbon atoms. Representative examplesof alkyl include, but are not limited to, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

The term “alkenyl” as used herein, means an unsaturated hydrocarbonchain containing from 2 to 20 carbon atoms and at least onecarbon-carbon double bond.

The term “alkynyl” as used herein, means an unsaturated hydrocarbonchain containing from 2 to 20 carbon atoms and at least onecarbon-carbon triple bond.

The term “alkoxyalkyl” as used herein, refers to an alkoxy group, asdefined herein, appended to the parent molecular moiety through analkylene group, as defined herein.

The term “arylalkyl” as used herein, refers to an aryl group, as definedherein, appended to the parent molecular moiety through an alkylenegroup, as defined herein.

The term “alkylene” as used herein, refers to a divalent group derivedfrom a straight or branched chain hydrocarbon of 1 to 10 carbon atoms,for example, of 2 to 5 carbon atoms. Representative examples of alkyleneinclude, but are not limited to, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,and —CH₂CH₂CH₂CH₂CH₂—.

The term “amide,” as used herein, means —C(O)NR— or —NRC(O)—, wherein Rmay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle,alkenyl, or heteroalkyl.

The term “aminoalkyl,” as used herein, means at least one amino group,as defined herein, is appended to the parent molecular moiety through analkylene group, as defined herein.

The term “amino” as used herein, means —NR_(x)R_(y), wherein R_(x) andR_(y) may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle,alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any othermoiety where amino appends together two other moieties, amino may be—NR_(x)—, wherein R_(x) may be hydrogen, alkyl, cycloalkyl, aryl,heteroaryl, heterocycle, alkenyl, or heteroalkyl. In some embodiments,amino is —NH₂.

The term “aryl” as used herein, refers to an aromatic group such as aphenyl group, or a bicyclic fused ring system. Bicyclic fused ringsystems are exemplified by a phenyl group appended to the parentmolecular moiety and fused to a cycloalkyl group, as defined herein, aphenyl group, a heteroaryl group, as defined herein, or a heterocycle,as defined herein. Representative examples of aryl include, but are notlimited to, indolyl, naphthyl, phenyl, quinolinyl, andtetrahydroquinolinyl.

The term “carboxyl” as used herein, means a carboxylic acid, or —COOH.

The term “cycloalkyl” means a monovalent saturated hydrocarbon ring orcarbocyclic group. The term “cycloalkenyl” means a monovalentunsaturated hydrocarbon ring, and cycloalkenyl groups include at leastone alkenyl. The term “cycloalkynyl” means a monovalent unsaturatedhydrocarbon ring, and cycloalkynyl groups include at least one alkynyl.Carbocyclic groups are monocyclic, or are fused, spiro, or bridgedbicyclic ring systems. Monocyclic carbocyclic groups contain 3 to 10carbon atoms, preferably 4 to 7 carbon atoms, and more preferably 5 to 6carbon atoms in the ring. Bicyclic carbocyclic groups contain 8 to 12carbon atoms, preferably 9 to 10 carbon atoms in the ring. Carbocyclicgroups may be substituted or unsubstituted. Cycloalkyl groups include,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexenyl, and cycloheptyl.

The term “haloalkyl” as used herein, means an alkyl group, as definedherein, in which one, two, three, four, five, six, seven, or eighthydrogen atoms are replaced by a halogen. Representative examples ofhaloalkyl include, but are not limited to, 2-fluoroethyl,2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl,and trifluoropropyl such as 3,3,3-trifluoropropyl.

The term “halogen” or “halo” as used herein, means Cl, Br, I, or F.

The term “heteroalkyl” as used herein, means an alkyl group, as definedherein, in which at least one of the carbons of the alkyl group isreplaced with a heteroatom, such as oxygen, nitrogen, and sulfur.Representative examples of heteroalkyls include, but are not limited to,alkyl ethers, secondary and tertiary alkyl amines, amides, and alkylsulfides.

The term “heteroaryl” as used herein, refers to an aromatic monocyclicring or an aromatic bicyclic ring system, including at least oneheteroatom, such as N, O, and S. The aromatic monocyclic rings are fiveor six membered rings containing at least one heteroatom independentlyselected from the group consisting of N, O, and S. The five memberedaromatic monocyclic rings have two double bonds, and the six memberedsix membered aromatic monocyclic rings have three double bonds. Thebicyclic heteroaryl groups are exemplified by a monocyclic heteroarylring appended to the parent molecular moiety and fused to a monocycliccycloalkyl group, as defined herein, a monocyclic aryl group, as definedherein, a monocyclic heteroaryl group, as defined herein, or amonocyclic heterocycle, as defined herein. Representative examples ofheteroaryl include, but are not limited to, indolyl, pyridinyl(including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl,pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, benzopyrazolyl,1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl,1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl,isothiazolyl, thienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl,oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl,quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolinyl,quinolinyl, 6,7-dihydro-1,3-benzothiazolyl, imidazo[1,2-a]pyridinyl,naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl,thiazolo[5,4-d]pyrimidin-2-yl.

The term “heterocycle” or “heterocyclic” as used herein means amonocyclic heterocycle, a bicyclic heterocycle (heterobicyclic), or atricyclic heterocycle. The monocyclic heterocycle is a three-, four-,five-, six-, seven-, or eight-membered ring containing at least oneheteroatom independently selected from the group consisting of O, N, andS. Heterocycloalkyl groups include carbon-carbon bonds that are allsingle bonds, heterocycloalkenyl groups include at least one alkenyl,and heterocycloalkynyl groups include at least one alkynyl. The three-or four-membered ring contains zero or one double bond, and oneheteroatom selected from the group consisting of O, N, and S. Thefive-membered ring contains zero or one double bond and one, two, orthree heteroatoms selected from the group consisting of O, N, and S. Thesix-membered ring contains zero, one, or two double bonds and one, two,or three heteroatoms selected from the group consisting of O, N, and S.The seven- and eight-membered rings contains zero, one, two, or threedouble bonds and one, two, or three heteroatoms selected from the groupconsisting of O, N, and S. Representative examples of monocyclicheterocycles include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl,piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl,pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl,thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclicheterocycle is a monocyclic heterocycle fused to a phenyl group, or amonocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclicheterocycle fused to a monocyclic cycloalkenyl, or a monocyclicheterocycle fused to a monocyclic heterocycle, or a bridged monocyclicheterocycle ring system in which two non-adjacent atoms of the ring arelinked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or analkenylene bridge of two, three, or four carbon atoms. Representativeexamples of bicyclic heterocycles include, but are not limited to,benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl,2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline,azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl),2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl,octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclicheterocycles are exemplified by a bicyclic heterocycle fused to a phenylgroup, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or abicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclicheterocycle fused to a monocyclic heterocycle, or a bicyclic heterocyclein which two non-adjacent atoms of the bicyclic ring are linked by analkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridgeof two, three, or four carbon atoms. Examples of tricyclic heterocyclesinclude, but not limited to, octahydro-2,5-epoxypentalene,hexahydro-2H-2,5-methanocyclopenta[b]furan,hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane(1-azatricyclo[3.3.1.1^(3,7)]decane), and oxa-adamantane(2-oxatricyclo[3.3.1.1^(3,7)]decane). The monocyclic, bicyclic, andtricyclic heterocycles are connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within the rings,and can be unsubstituted or substituted.

The term “heteroarylalkyl” as used herein, refers to a heteroaryl group,as defined herein, appended to the parent molecular moiety through analkylene group, as defined herein.

The term “heterocycloalkyl” as used herein, refers to a heterocyclegroup, as defined herein, appended to the parent molecular moietythrough an alkylene group, as defined herein.

The term “hydroxyl” or “hydroxy” as used herein, means an —OH group.

The term “hydroxyalkyl” as used herein, means at least one —OH group, isappended to the parent molecular moiety through an alkylene group, asdefined herein.

The term “nitro” means a —NO₂ group.

In some instances, the number of carbon atoms in a hydrocarbylsubstituent (e.g., alkyl or cycloalkyl) is indicated by the prefix“C_(x)-C_(y)-”, wherein x is the minimum and y is the maximum number ofcarbon atoms in the substituent. Thus, for example, “C₁-C₃ alkyl” refersto an alkyl substituent containing from 1 to 3 carbon atoms.

The term “substituted” refers to a group that may be further substitutedwith one or more non-hydrogen substituent groups. Substituent groupsinclude, but are not limited to, halogen, ═O (oxo), ═S (thioxo), cyano,nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl,aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl,arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene,aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl,arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl,arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate,and acyl.

For compounds described herein, groups and substituents thereof may beselected in accordance with permitted valence of the atoms and thesubstituents, such that the selections and substitutions result in astable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

The term “administration” or “administering,” as used herein, refers toproviding, contacting, and/or delivery of a compound or agent by anyappropriate route to achieve the desired effect. These compounds oragents may be administered to a subject in numerous ways including, butnot limited to, orally, ocularly, nasally, intravenously, topically, asaerosols, suppository, etc. and may be used in combination.

The term “agonist” refers to a biologically active ligand that binds toits complementary biologically active receptor and activates thereceptor either to cause a biological response in the receptor or toenhance a biological activity of the receptor. An agonist may trigger(e.g., initiate or promote), partially or fully enhance, stimulate, oractivate one or more biological activities. An agonist may mimic theaction of a naturally occurring substance. An agonist of G-CSF-R inducesthe same intracellular biological response as those triggered by G-CSF.

The term “antagonist” means an agent that inhibits the effect of anagonist. The term “antagonist” may also refer to a molecule which blocks(e.g., reduces or prevents) a biological activity.

The terms “control,” “reference level,” and “reference” are used hereininterchangeably. The reference level may be a predetermined value orrange, which is employed as a benchmark against which to assess themeasured result. “Control group” as used herein refers to a group ofcontrol subjects. The predetermined level may be a cutoff value from acontrol group. The predetermined level may be an average from a controlgroup. Cutoff values (or predetermined cutoff values) may be determinedby Adaptive Index Model (AIM) methodology. Cutoff values (orpredetermined cutoff values) may be determined by a receiver operatingcurve (ROC) analysis from biological samples of the patient group. ROCanalysis, as generally known in the biological arts, is a determinationof the ability of a test to discriminate one condition from another,e.g., to determine the performance of each marker in identifying apatient having CRC. A description of ROC analysis is provided in P. J.Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of whichis hereby incorporated by reference in its entirety. Alternatively,cutoff values may be determined by a quartile analysis of biologicalsamples of a patient group. For example, a cutoff value may bedetermined by selecting a value that corresponds to any value in the25th-75th percentile range, preferably a value that corresponds to the25th percentile, the 50th percentile or the 75th percentile, and morepreferably the 75th percentile. Such statistical analyses may beperformed using any method known in the art and can be implementedthrough any number of commercially available software packages (e.g.,from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station,Tex.; SAS Institute Inc., Cary, N.C.). The healthy or normal levels orranges for a target or for a protein activity may be defined inaccordance with standard practice. A control may be a subject, or asample therefrom, whose disease state is known. The subject, or sampletherefrom, may be healthy, diseased, diseased prior to treatment,diseased during treatment, diseased after treatment, or healthy aftertreatment, or a combination thereof. The term “normal subject” as usedherein means a healthy subject, i.e. a subject having no clinical signsor symptoms of disease. The normal subject is clinically evaluated forotherwise undetected signs or symptoms of disease, which evaluation mayinclude routine physical examination and/or laboratory testing. In someembodiments, the control is a healthy control. In some embodiments, thecontrol comprises neurodegenerative disease.

The term “effective dosage” or “therapeutic dosage” as used herein meansa dosage of a drug effective for periods of time necessary, to achievethe desired therapeutic result. An effective dosage may be determined bya person skilled in the art and may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the drug to elicit a desired response in the individual.

The terms “inhibit” or “inhibiting” mean that an activity is decreasedor prevented in the presence of an inhibitor as opposed to in theabsence of the inhibitor. The term “inhibition” refers to the reductionor down regulation of a process or the elimination of a stimulus for aprocess, which results in the absence or minimization of the expressionor activity of a biomarker or polypeptide. Inhibition may be direct orindirect. Inhibition may be specific, that is, the inhibitor inhibits abiomarker or polypeptide and not others.

“Neurodegenerative Diseases” are disorders characterized by, resultingfrom, or resulting in the progressive loss of structure or function ofneurons, including death of neurons. Neurodegeneration can be found inmany different levels of neuronal circuitry ranging from molecular tosystemic. Some neurodegenerative diseases are caused by geneticmutations. Some neurodegenerative diseases are classified asproteopathies because they are associated with the aggregation ofmisfolded proteins. Neurodegenerative diseases include, for example,Alzheimer's Disease (AD), amyotrophic lateral sclerosis (ALS),Parkinson's Disease (PD), prion disease, motor neuron disease,Huntington's Disease, spinocerebellar ataxia, and spinal muscularatrophy.

“Polynucleotide” as used herein can be single stranded or doublestranded, or can contain portions of both double stranded and singlestranded sequence. The polynucleotide can be nucleic acid, natural orsynthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where thepolynucleotide can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,and isoguanine. Polynucleotides can be obtained by chemical synthesismethods or by recombinant methods.

A “peptide” or “polypeptide” is a linked sequence of two or more aminoacids linked by peptide bonds. The polypeptide can be natural,synthetic, or a modification or combination of natural and synthetic.Peptides and polypeptides include proteins such as binding proteins,receptors, and antibodies. The terms “polypeptide”, “protein,” and“peptide” are used interchangeably herein. “Primary structure” refers tothe amino acid sequence of a particular peptide. “Secondary structure”refers to locally ordered, three dimensional structures within apolypeptide. These structures are commonly known as domains, e.g.,enzymatic domains, extracellular domains, transmembrane domains, poredomains, and cytoplasmic tail domains. Domains are portions of apolypeptide that form a compact unit of the polypeptide and aretypically 15 to 350 amino acids long. Exemplary domains include domainswith enzymatic activity or ligand binding activity. Typical domains aremade up of sections of lesser organization such as stretches ofbeta-sheet and alpha-helices. “Tertiary structure” refers to thecomplete three dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.

“Recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid,protein, or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

“Sample” or “test sample” as used herein can mean any sample in whichthe presence and/or level of a biomarker or target is to be detected ordetermined. Samples may include liquids, solutions, emulsions, mixtures,or suspensions. Samples may include a medical sample. Samples mayinclude any biological fluid or tissue, such as blood, whole blood,fractions of blood such as plasma and serum, peripheral bloodmononuclear cells (PBMCs), muscle, interstitial fluid, sweat, saliva,urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasalsecretions, sputum, amniotic fluid, bronchoalveolar lavage fluid,gastric lavage, emesis, fecal matter, lung tissue, peripheral bloodmononuclear cells, total white blood cells, lymph node cells, spleencells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid,skin, or combinations thereof. In some embodiments, the sample comprisesan aliquot. In other embodiments, the sample comprises a biologicalfluid. Samples can be obtained by any means known in the art. The samplecan be used directly as obtained from a patient or can be pre-treated,such as by filtration, distillation, extraction, concentration,centrifugation, inactivation of interfering components, addition ofreagents, and the like, to modify the character of the sample in somemanner as discussed herein or otherwise as is known in the art. Samplesmay be obtained before treatment, before diagnosis, during treatment,after treatment, or after diagnosis, or a combination thereof.

The term “specificity” as used herein refers to the number of truenegatives divided by the number of true negatives plus the number offalse positives, where specificity (“spec”) may be within the range of0<spec<1. Hence, a method that has both sensitivity and specificityequaling one, or 100%, is preferred.

By “specifically binds,” it is generally meant that a compound orpolypeptide binds to a target when it binds to that target more readilythan it would bind to a random, unrelated target.

“Subject” as used herein can mean a mammal that wants or is in need ofthe herein described compounds or methods. The subject may be a human ora non-human animal. The subject may be a mammal. The mammal may be aprimate or a non-primate. The mammal can be a primate such as a human; anon-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat,camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-humanprimate such as, for example, monkey, chimpanzee, gorilla, orangutan,and gibbon. The subject may be of any age or stage of development, suchas, for example, an adult, an adolescent, or an infant.

“Traumatic brain injury” or “TBI” refers to insult to the brain causedby an external physical force that may produce a diminished or alteredstate of consciousness and that results in an impairment of cognitiveabilities or physical functioning. The pathology of TBI may includethree phases: (1) primary injury to brain tissue and/or the cerebralvasculature; (2) the secondary injury, which includes physiological,neuroinflammatory, and biochemical processes triggered by the primaryinsult; and (3) regenerative responses, including enhanced proliferationof neural progenitor cells and endothelial cells. Brain regions include,for example, hippocampus, cortex, striatum, and corpus callosum.

The terms “treat,” “treated,” or “treating” as used herein refers to atherapeutic wherein the object is to slow down (lessen) an undesiredphysiological condition, disorder or disease, or to obtain beneficial ordesired clinical results. For the purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationof symptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment.

“Variant” as used herein with respect to a polynucleotide means (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a polynucleotide that is substantially identical to a referencedpolynucleotide or the complement thereof; or (iv) a polynucleotide thathybridizes under stringent conditions to the referenced polynucleotide,complement thereof, or a sequences substantially identical thereto.

A “variant” can further be defined as a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Representative examples of “biological activity”include the ability to be bound by a specific antibody or polypeptide orto promote an immune response. Variant can mean a substantiallyidentical sequence. Variant can mean a functional fragment thereof.Variant can also mean multiple copies of a polypeptide. The multiplecopies can be in tandem or separated by a linker. Variant can also meana polypeptide with an amino acid sequence that is substantiallyidentical to a referenced polypeptide with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids. See Kyte et al., J. Mol. Biol.1982, 157, 105-132. The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indices of ±2 are substituted. The hydrophobicity of aminoacids can also be used to reveal substitutions that would result inpolypeptides retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a polypeptide permitscalculation of the greatest local average hydrophilicity of thatpolypeptide, a useful measure that has been reported to correlate wellwith antigenicity and immunogenicity, as discussed in U.S. Pat. No.4,554,101, which is fully incorporated herein by reference. Substitutionof amino acids having similar hydrophilicity values can result inpolypeptides retaining biological activity, for example immunogenicity,as is understood in the art. Substitutions can be performed with aminoacids having hydrophilicity values within ±2 of each other. Both thehydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. A variant can be a polynucleotide sequence that issubstantially identical over the full length of the full gene sequenceor a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical over the full length of the genesequence or a fragment thereof. A variant can be an amino acid sequencethat is substantially identical over the full length of the amino acidsequence or fragment thereof. The amino acid sequence can be 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical over the full length of the aminoacid sequence or a fragment thereof. In some embodiments, variantsinclude homologues. Homologues may be polynucleotides or polypeptides orgenes inherited in two species by a common ancestor.

2. Granulocyte Colony Stimulating Factor (G-CSF)

Granulocyte-colony stimulating factor (G-CSF or GCSF) is also known ascolony-stimulating factor 3 (CSF 3). G-CSF is a glycoprotein that mayact as a cytokine and/or hormone, as a type of colony-stimulatingfactor. G-CSF may be produced by a number of different tissues such as,for example, endothelium, macrophages, and other immune cells.

G-CSF may comprise a polypeptide of SEQ ID NO: 1. G-CSF polypeptide maybe encoded by a polynucleotide of SEQ ID NO: 2. Commercially availableforms of G-CSF include filgrastim and lenograstim. Commerciallyavailable filgrastim includes ZARXIO® (Novartis, Basel, Switzerland),GRANIX® (Teva Pharmaceuticals Industries Ltd., Petah Tikva, Israel), andNEUPOGEN® (Amgen Inc., Thousand Oaks, Calif.). Commercially availablelenograstim includes GRANOCYTE™ (Chugai Pharmaceutical Co., Tokyo,Japan).

G-CSF binds the G-CSF receptor (G-CSF-R) to elicit its effects.

a. Granulocyte Colony Stimulating Factor Receptor (G-CSF-R)

Granulocyte-colony stimulating factor receptor (G-CSF-R) is a cellsurface receptor and binds G-CSF. G-CSF-R may comprise a polypeptide ofSEQ ID NO: 3. G-CSF-R may be found, for example, on the cell surface ofprecursor cells in the bone marrow, and on the cell surface of neuronsin the brain and spinal cord. G-CSF-R may be found in the brain in theneurogenic zone of the hippocampus, the sub-ventricular zone, theolfactory bulb, pyramidal cells in corticol layers, entorhinal cortex,and/or Purkinje cells of the cerebellum. G-CSF-R may also be found onthe surfaces of endothelial cells, lymphocytes, platelets, and/orneutrophils. In some embodiments, G-CSF-R is from human. In someembodiments, G-CSF-R is from mouse. G-CSF-R has a composite structureconsisting of an immunoglobulin-like (Ig) domain, a cytokine-receptorhomologous (CRH) domain, and three fibronectin type III (FNIII) domainsin the extracellular region. The CRH region of G-CSF-R is a domain forligand binding and for mediating the signal.

Binding of G-CSF to G-CSF-R triggers homodimerization of the receptorand activation of complex signal transduction and anti-apoptoticpathways. Upon binding G-CSF-R, the effects of G-CSF include peripheraland central activities. Central activities of G-CSF are those in thecentral nervous system (CNS), such as, for example, inducingneurogenesis to increase neuroplasticity and to counteract apoptosis,serving as a neurotrophic factor, promoting neuronal survival,stimulating neural stem cell and/or progenitor cell proliferation in,for example, the hippocampus. Peripheral activities of G-CSF are thosebeyond the CNS, such as, for example, stimulating or increasinghematopoiesis and increasing levels of polymorphonucleocytes, increasingblood stem cells, and increasing circulating monocytes. The beneficialeffects of G-CSF in the treatment of diseases or conditions may resultfrom its central activities in the brain, rather than its peripheraleffects. Binding of G-CSF to G-CSF-R may stimulate the bone marrow toproduce granulocytes and stem cells and release them into thebloodstream; stimulate the survival, proliferation, differentiation, andfunction of neutrophil precursors and mature neutrophils; increaseproliferation of neural stem/progenitor cells; promote or increaseneurogenesis (the generation of new neurons in the brain) or enhancesurvival of new neurons in, for example, the hippocampus; promote brainrepair and improve cognitive performance after TBI; increase thegeneration of white blood cells; increase the generation ofhematopoietic stem/progenitor cells; activate astrocytes and/ormicroglia; increase levels of neurotrophic factors such as glial cellline derived neurotrophic factor (GDNF) and/or brain derivedneurotrophic factor (BDNF); increase the expression of PKCδ isoforms;increase the expression of STAT3; increase the expression of Bcl-2;and/or decrease the expression of Bax; or any combination thereof.

a. Protein Kinase C Delta VIII (PKCδVIII)

Protein Kinase C Delta (PKCδ) is a serine/threonine kinase mediatingcellular growth, differentiation, and apoptosis. PKCδ is alternativelyspliced to generate isoforms with distinct functions in apoptosis. MousePKCδ has two alternatively spliced variants—PKCδI and PKCδII—which haveopposite functions in cell proliferation, differentiation, and apoptosis(mouse PKCδII promotes survival while mouse PKCδI promotes apoptosis).Mouse PKCδII is generated by utilization of an alternative downstream 5′splice site of PKCδ pre-mRNA exon 9. Mouse PKCδII is resistant tocleavage by caspase-3 and may be associated with and promoteneurogenesis and neuronal differentiation. PKCδVIII is the human homologof mouse PKCδII. Human PKCδVIII is an anti-apoptotic protein.

b. Signal Transducer and Activator of Transcription 3 (STAT3)

Signal transducer and activator of transcription 3 (STAT3) is a proteinand transcription factor. In response to cytokines and growth factors,STAT3 is phosphorylated by receptor-associated Janus kinases (JAK),forms homo- or heterodimers, and translocates to the cell nucleus whereit acts as a transcription factor. STAT3 mediates the expression of avariety of genes in response to cell stimuli and plays a key role inmany cellular processes such as cell growth and apoptosis.

c. B-Cell Lymphoma 2 (Bcl2)

Upon binding G-CSF-R, G-CSF may act as a neurotrophic factor toupregulate the expression of Bcl2. Bcl2 is a protein localized to theouter membrane of mitochondria and inhibits apoptosis. Activation of themitochondrial pathway of apoptosis culminates in mitochondrial outermembrane permeabilization and cell death. BAX and BAK are proteins thatdisrupt the mitochondrial membrane, and the antiapoptotic protein Bcl2constrains or inhibits BAX and BAK.

d. Bax

BAX is a protein that regulates apoptosis and is also known asBcl-2-like protein 4. BAX is found in the cytosol or associated withorganelle membranes. BAX may interact with, and increase the opening of,the mitochondrial voltage-dependent anion channel (VDAC), which leads tothe loss in membrane potential and the release of cytochrome c.Alternatively, BAX may help form an oligomeric pore in the mitochondrialmembrane. The action of BAX on the mitochondrial membrane culminates inmitochondrial outer membrane permeabilization and cell death.

3. Compounds

Detailed herein are compounds that modulate the activity of G-CSF and/orG-CSF-R. The compound may be according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring;

R³ and R⁴ are each hydrogen, or R³ and R⁴ together with the atoms towhich they are attached form a six-membered heterocyclic ring, whereinthe ring is unsubstituted or substituted with one substituent selectedfrom amino, nitro, methyl, ethyl, and hydroxyl;

X is —C(R⁵)(R⁶)-phenyl wherein the phenyl is substituted with R⁷ and R⁸,or X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰;

R⁵ and R⁶ are each independently selected from hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, or R⁵ and R⁶ together form an oxo group;

R⁷ is hydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy;

R⁸ is halogen, hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy;

each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄alkoxy, amino, or nitro; and

each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro.

In some embodiments, R¹ and R² are each hydrogen. In some embodiments,R¹ and R² together with the carbon atoms to which they are attached forma phenyl ring.

In some embodiments, R³ and R⁴ are each hydrogen. In some embodiments,R³ and R⁴ together with the atoms to which they are attached form asix-membered heterocyclic ring, wherein the ring is unsubstituted orsubstituted with one substituent selected from amino, nitro, methyl,ethyl, or hydroxyl. In some embodiments, the six-membered heterocyclicring is substituted with amino. In some embodiments, the six-memberedheterocyclic ring is substituted with nitro.

In some embodiments, X is —C(R⁵)(R⁶)-phenyl wherein the phenyl issubstituted with R⁷ and R⁸. In some embodiments, R⁵ and R⁶ are eachindependently hydroxyl or methyl. In some embodiments, R⁵ and R⁶together form an oxo group. In some embodiments, R⁷ is hydroxyl oralkoxy. In some embodiments, R⁷ is hydroxyl. In some embodiments, R⁸ ishalogen. In some embodiments, R⁸ is halogen, and halogen is Cl.

In some embodiments, X is indole substituted with one or two R⁹. In someembodiments, each R⁹ is independently halogen or methoxy.

In some embodiments, X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰. In some embodiments, eachR¹⁰ is independently hydroxyl, methoxy, or nitro. In some embodiments,the phenyl is substituted with 0 or 1 R¹⁰.

In some embodiments, the compound is of Formula I, wherein R¹ and R² areeach hydrogen, R³ and R⁴ are each hydrogen, X is —C(R⁵)(R⁶)-phenylwherein the phenyl is substituted with R⁷ and R⁸, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₇ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R² areeach hydrogen, R³ and R⁴ are each hydrogen, X is —C(R⁵)(R⁶)-phenylwherein the phenyl is substituted with R⁷ and R⁸, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₈ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R² areeach hydrogen, R³ and R⁴ are each hydrogen, X is —C(R⁵)(R⁶)-phenylwherein the phenyl is substituted with R⁷ and R⁸, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₉ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R² areeach hydrogen, R³ and R⁴ are each hydrogen, X is —C(R⁵)(R⁶)-phenylwherein the phenyl is substituted with R⁷ and R⁸, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is hydroxyl, and R⁸ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R² areeach hydrogen, R³ and R⁴ are each hydrogen, X is —C(R⁵)(R⁶)-phenylwherein the phenyl is substituted with R⁷ and R⁸, wherein R⁵ and R⁶together form an oxo group, R⁷ is hydroxyl, and R⁸ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R²together with the carbon atoms to which they are attached form a phenylring, R³ and R⁴ together with the atoms to which they are attached forma six-membered heterocyclic ring wherein the ring is substituted withamino, and X is indole substituted with one R⁹, wherein R⁹ is halogen.

In some embodiments, the compound is of Formula I, wherein R¹ and R²together with the carbon atoms to which they are attached form a phenylring, R³ and R⁴ together with the atoms to which they are attached forma six-membered heterocyclic ring wherein the ring is substituted withamino, and X is indole substituted with one R⁹, wherein R⁹ is methoxy.

In some embodiments, the compound is of Formula I, wherein R¹ and R²together with the carbon atoms to which they are attached form a phenylring, R³ and R⁴ together with the atoms to which they are attached forma six-membered heterocyclic ring wherein the ring is substituted withamino, and X is pyrazole substituted with phenyl wherein the phenyl issubstituted with one R¹⁰, wherein R¹⁰ is hydroxyl.

In some embodiments, the compound is of Formula I, wherein R¹ and R²together with the carbon atoms to which they are attached form a phenylring, R³ and R⁴ together with the atoms to which they are attached forma six-membered heterocyclic ring wherein the ring is substituted withamino, and X is pyrazole substituted with phenyl wherein the phenyl issubstituted with one R¹⁰, wherein R¹⁰ is methoxy.

In some embodiments, the compound is of Formula I, wherein R¹ and R²together with the carbon atoms to which they are attached form a phenylring, R³ and R⁴ together with the atoms to which they are attached forma six-membered heterocyclic ring wherein the ring is substituted withamino, and X is pyrazole substituted with phenyl wherein the phenyl issubstituted with one R¹⁰, wherein R¹⁰ is nitro.

In some embodiments, the compound is according to Formula II:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰;

each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄alkoxy, amino, or nitro; and

each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro.

In some embodiments, X is indole substituted with one R⁹. In someembodiments, R⁹ is halogen or methoxy.

In some embodiments, X is pyrazole substituted with phenyl, wherein thephenyl is substituted with one R¹⁰. In some embodiments, R¹⁰ ishydroxyl, methoxy, or nitro.

In some embodiments, the compound is of Formula II, wherein X is indolesubstituted with one R⁹, wherein R⁹ is halogen.

In some embodiments, the compound is of Formula II, wherein X is indolesubstituted with one R⁹, wherein R⁹ is methoxy.

In some embodiments, the compound is of Formula II, wherein X ispyrazole substituted with phenyl, wherein the phenyl is substituted withone R¹⁰, wherein R¹⁰ is hydroxyl.

In some embodiments, the compound is of Formula II, wherein X ispyrazole substituted with phenyl, wherein the phenyl is substituted withone R¹⁰, wherein R¹⁰ is methoxy.

In some embodiments, the compound is of Formula II, wherein X ispyrazole substituted with phenyl, wherein the phenyl is substituted withone R¹⁰, wherein R¹⁰ is nitro.

In some embodiments, the compound is according to Formula III:

wherein R⁵ and R⁶ are each independently hydrogen, hydroxyl, methyl,ethyl, methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group;

R⁷ is hydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and

R⁸ is halogen, hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy.

In some embodiments, R⁵ and R⁶ are each independently hydroxyl ormethyl. In some embodiments, R⁵ and R⁶ together form an oxo group. Insome embodiments, R⁷ is hydroxyl or alkoxy. In some embodiments, R⁷ ishydroxyl. In some embodiments, R⁸ is halogen. In some embodiments, R⁸ ishalogen, and halogen is Cl.

In some embodiments, the compound is of Formula III, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₇ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula III, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₈ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula III, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is C₉ alkoxy, and R⁸ is halogen.

In some embodiments, the compound is of Formula III, wherein R⁵ ishydroxyl, R⁶ is methyl, R⁷ is hydroxyl, and R⁸ is halogen.

In some embodiments, the compound is of Formula III, wherein R⁵ and R⁶together form an oxo group, R⁷ is hydroxyl, and R⁸ is halogen.

In some embodiments, the compound is selected from the following:

In some embodiments, the compound is selected from the following:

In some embodiments, the compound is selected from the following:

In some embodiments, the compound is selected from the followingcompounds:

In some embodiments, the compound excludes or is not one of thefollowing compounds:

In some embodiments, the compound excludes or is not one of thefollowing compounds:

a. Synthesis of Compounds

Compounds 1-5 were obtained from Moffitt Cancer Center Chemistry unit(Tampa, Fla.). Alternatively, compounds 1-5 may be synthetically made bymethods known to one of skill in the art. Compounds 6-10 arecommercially available, for example, from Chembridge Corporation (SanDiego, Calif.). Alternatively, compounds 6-10 may be synthetically madeby methods known to one of skill in the art.

b. Effects of Compounds

The compounds as detailed herein may act as agonists, antagonists, orpartial agonists/antagonists of G-CSF-R. In some embodiments, thecompound modulates the effects of G-CSF-R. In some embodiments, thecompound modulates the effects of G-CSF. In some embodiments, thecompound binds G-CSF-R. In some embodiments, the compound binds G-CSF-Rwith greater affinity than G-CSF. In some embodiments, the compoundbinds G-CSF-R with less affinity than G-CSF. In some embodiments, thecompound displaces G-CSF from G-CSF-R. The compound may displace atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofG-CSF from G-CSF-R. In some embodiments, the compound displaces at least50% of G-CSF from the G-CSF receptor. In some embodiments, the compounddisplaces at least 75% of G-CSF from the G-CSF receptor.

In some embodiments, the compound is an agonist of G-CSF-R. The compoundmay trigger signal transduction pathways and effects similar to theeffects of the natural ligand G-CSF, for example, those as detailedherein. The compound may mimic the neurotrophic and/or immune-modulatingactions of G-CSF. In some embodiments, the compound is a central agonistof G-CSF-R. In such embodiments, the compound triggers effects similarto those of the natural ligand G-CSF in the CNS, but not those outsidethe CNS. In some embodiments, the compound is a peripheral agonist ofG-CSF-R. In such embodiments, the compound triggers effects similar tothose of the natural ligand G-CSF outside the central nervous system,but not those in the CNS.

The compound, upon administration to a subject, may elicit a variety ofeffects as an agonist of G-CSF-R. In some embodiments, the compounddecreases amyloid burden, enhances neurogenesis, enhancessynaptogenesis, enhances cognitive performance, increases expression ofBcl2, increases expression of PKCδVIII, increases expression of STAT3,decreases expression of Bax, or any combination thereof. In someembodiments, the compound increases expression of Bcl2. In someembodiments, the compound increases expression of PKCδVIII. In someembodiments, the compound increases expression of STAT3. In someembodiments, the compound decreases expression of Bax.

In some embodiments, the compound is a central antagonist of G-CSF-R. Insuch embodiments, the compound inhibits, or does not trigger, effectssimilar to those of the natural ligand G-CSF in the CNS, but has littleto no activity outside the CNS. In some embodiments, the compound is aperipheral antagonist of G-CSF-R. In such embodiments, the compoundinhibits, or does not trigger, effects similar to those of the naturalligand G-CSF outside the CNS, but has little to no activity in the CNS.In some embodiments, the compound has a minimal effect on leukopoiesis.

In some embodiments, the compound may act as a partial or mixedagonist/antagonist of G-CSF-R. In such embodiments, the compound elicitsa combination of the agonist and antagonist effects detailed above. Forexample, the compound may be an antagonist of G-CSF biological responsesin monocytes, but not neurons. The compound may be a central agonist anda peripheral antagonist.

The activity of the compound may be examined by measuring the capacityof the compound to displace labelled G-CSF from G-CSF-R expressed incell lines. Suitable labels are known in the art, such as, for example,radiolabels. The cell line may include, for example, THP-1 (which is ahuman monocyte cell line representative of the peripheral immunesystem), and SH-SY5Y (which is a human neuroblastoma cell line). Thecompounds as detailed herein may displace labelled G-CSF from G-CSF-Rexpressed in cell lines.

The activity of the compound may be examined by measuring the expressionof, for example, Bcl2 and/or PKCδVIII protein, in a cell line. The cellline may include, for example, THP-1 or SH-SY5Y. Bcl2 and/or PKCδVIIIprotein may be measured by a method according to one of skill in theart, such as, for example, immunoassays, Western blot, and probes tomRNA.

The activity of the compound may be examined by administering thecompound to mice before, concomitantly, or after administeringcontrolled corticol impact (CCI, an experimental TBI) to the mice, andthen testing the mice in assays such as radial arm water maze (RAWM; ahippocampal-dependent spatial learning task that does not rely onlocomotor ability or swimming speed), testing motor balance andcoordination on a rotating cylinder (rotarod), measuring microglial andastroglial response, measuring hippocampal neurogenesis, measuringneurotropic factors such as brain-derived neurotrophic factor (BDNF) andglial cell line-derived neurotrophic factor (GDNF), and/or measuringcytokines in brain homogenates. Brain regions include, for example,hippocampus, cortex, striatum, and corpus callosum. The neurotropicfactors and cytokines may be measured by any means known in the art suchas, for example, immunohistochemistry. Astroglial and microglialresponse may be monitored by any means known in the art such as, forexample, immunostaining with antibodies to GFAP and Iba-1, respectively.Neurogenesis may be measured, for example, by measuring the expressionof doublecortin (DCX; a microtubule-associated protein and a marker ofimmature neurons) using an antibody to DCX. The central action of thecompound in the CNS may be examined by comparing the effects of thecompound with and without the presence of a selective chemokine receptorantagonist of monocyte chemoattractant protein-1 (MCP-1). In suchembodiments, an MCP-1 inhibitor may be used to decrease the infiltrationof bone marrow-derived cells (BMDC) such as monocytes into the CNS, tofacilitate isolation of the peripheral action of recruiting monocytesfrom the blood into the brain from the central actions in the CNS. MCP-1mediates recruitment of inflammatory cells to sites of tissue injury.Selective MCP-1 inhibitors include, for example, C—C motif receptor 2(CCR2) antagonists. CCR2 is a G-protein-coupled receptor that binds itsligand MCP-1. CCR2 antagonists include, for example, RS504393(6-methyl-10-[2-(5-methyl-2-phenyl-4-oxazolyl)ethyl]-spiro[4H-3,1-benzoxazine-4,40-piperidin]-2(1H)-one),which may be purchased commercially from, for example, Tocris BiosciInc. (Minneapolis, Minn.). In some embodiments, the compound improvesrecovery in mice after CCI. In some embodiments, the direct actions ofthe compound in the CNS improve recovery in mice after CCI. In someembodiments, the compound improves motor function, such as, for example,after injury. In some embodiments, the compound improves spatiallearning, such as, for example, after injury. In some embodiments, thecompound improves performance in RAWM and/or rotarod after CCI. In someembodiments, the compound activates astrocytes and/or microglia. In someembodiments, the compound increases levels of GDNF and/or BDNF. In someembodiments, the compound increases expression of DCX and/or increasesthe number of cells expressing DCX. In some embodiments, the compoundincreases expression of GFAP. In some embodiments, the compoundincreases expression of Iba-1.

In some embodiments, the compound is co-administered with a G-CSFpolypeptide, or in some embodiments, a polynucleotide encoding a G-CSFpolypeptide.

In some embodiments, the compound treats disorders including, but notlimited to, neurodegenerative disease, stroke, traumatic brain injury(TBI), impaired motor function, and impaired cognitive function, or anycombination thereof.

4. Administration

A composition may comprise the compound detailed above. The compoundscan be formulated into a composition in accordance with standardtechniques well known to those skilled in the pharmaceutical art. Thecomposition may be prepared for administration to a subject. Suchcompositions can be administered in dosages and by techniques well knownto those skilled in the medical arts taking into consideration suchfactors as the age, sex, weight, and condition of the particularsubject, and the route of administration.

The compound can be administered prophylactically or therapeutically. Inprophylactic administration, the compound can be administered in anamount sufficient to induce a response. In therapeutic applications, thecompounds are administered to a subject in need thereof in an amountsufficient to elicit a therapeutic effect. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on, e.g., the particular compositionof the compound regimen administered, the manner of administration, thestage and severity of the disease, the general state of health of thepatient, and the judgment of the prescribing physician.

The compound can be administered by methods well known in the art asdescribed in Donnelly et al. (Ann. Rev. Immunol. 1997, 15, 617-648);Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of whichare incorporated herein by reference in their entirety. The compound canbe complexed to particles or beads that can be administered to anindividual, for example, using a vaccine gun. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration.

The compound can be delivered via a variety of routes. Typical deliveryroutes include parenteral administration, e.g., intradermal,intramuscular or subcutaneous delivery. Other routes include oraladministration, intranasal, intravaginal, transdermal, intravenous,intraarterial, intratumoral, intraperitoneal, and epidermal routes. Insome embodiments, the compound is administered intravenously,intraarterially, or intraperitoneally to the subject.

The compound can be a liquid preparation such as a suspension, syrup, orelixir. The compound can be incorporated into liposomes, microspheres,or other polymer matrices (such as by a method described in Felgner etal., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein byreference in their entirety). Liposomes can consist of phospholipids orother lipids, and can be nontoxic, physiologically acceptable andmetabolizable carriers that are relatively simple to make andadminister.

The compound may be used as a vaccine. The vaccine can be administeredvia electroporation, such as by a method described in U.S. Pat. No.7,664,545, the contents of which are incorporated herein by reference.The electroporation can be by a method and/or apparatus described inU.S. Pat. Nos. 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034;6,208,893; 6,192,270; 6,181,964; 6,150,148; 6,120,493; 6,096,020;6,068,650; and 5,702,359, the contents of which are incorporated hereinby reference in their entirety. The electroporation can be carried outvia a minimally invasive device.

In some embodiments, the compound is administered in a controlledrelease formulation. The compound may be released into the circulation,for example. In some embodiments, the compound may be released over aperiod of at least about 1 day, at least about 2 days, at least about 3days, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 1 week, at least about 1.5weeks, at least about 2 weeks, at least about 2.5 weeks, at least about3.5 weeks, at least about 4 weeks, or at least about 1 month.

5. Methods

a. Methods of Treating a Condition

Provided herein are methods of treating a condition in a subject. Themethod may include administering to a subject a compound as detailedherein. In some embodiments, the condition is selected from the groupconsisting of neurodegenerative disease, stroke, traumatic brain injury(TBI), impaired motor function, and impaired cognitive function. In someembodiments, the neurodegenerative disease is selected from the groupconsisting of Alzheimer's Disease (AD), amyotrophic lateral sclerosis(ALS), Parkinson's Disease (PD), prion disease, motor neuron disease,Huntington's Disease, spinocerebellar ataxia, and spinal muscularatrophy.

b. Methods of Stimulating the Central Nervous System G-CSF-R

Provided herein are methods of stimulating the central nervous systemG-CSF-R. The method may include administering to a subject a compound asdetailed herein.

6. Examples Examples Example 1 Materials and Methods

Cell Cultures.

Two human cell lines were chosen for cell culture studies. One was ahuman monocyte cell line that is representative of the peripheral immunesystem, THP-1, which grows in suspension. The other cell line was ahuman neuroblastoma cell line (SH-SY5Y) which is a mixed cell linealthough only adherent cells were utilized (Xie, H. R., et al. Chin.Med. J. (Engl) 2010, 123, 1086-92). Both cell lines are known to expressthe G-CSF-R. Both cell lines were purchased from ATCC (Manassas, Va.).THP-1 cells were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum (FBS) plus 1% penicillin-streptomycin (ATCC,Manassas, Va.) in a humidified atmosphere of 5% CO₂ at 37° C. SH-SY5Ycell line were cultured in 1:1 EMEM/F12 medium with 10% FBS plus 1%penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37° C.

In Silico Modeling of G-CSF Receptor to Identify Potential G-CSFMimetics or Antagonists.

Compounds 1-3 (TABLE 1) were originally discovered by Kusano et al.(Blood 2004, 103, 836-42) and found to stimulate blood stem cellproliferation and to increase levels of circulating leukocytes in arodent model. The other drugs were discovered using an initial screenbased on in silico modeling of the G-CSF receptor (GCSF-R). Schödinger3D-modeling software (https://www.schrodinger.com/smdd/) was used toimport a molecular model of G-CSF bound to its receptor from the NCBIprotein database (http://www.ncbi.nlm.nih.gov/protein/1PGR_C). G-CSF-Rhas previously been cloned, purified, and crystallized to generate X-raydiffraction patterns to allow reconstruction and visualization in 3Dcomputer models. G-CSF-R has a composite structure consisting of animmunoglobulin-like (Ig) domain, a cytokine-receptor homologous (CRH)domain, and three fibronectin type III (FNIII) domains in theextracellular region. The CRH region of G-CSF-R is an essential domainfor ligand binding and mediating the signal. G-CSF-R dimerizationinduced by G-CSF binding has been demonstrated to be a common signaltransduction mechanism.

The goal in the modeling study was to find small molecules that fit thesite(s) on the G-CSF-R where binding with its natural ligand G-CSFoccurs. After modeling the first 3 molecules identified as potentialG-CSF mimetics, other molecules were screened and selected for furtherstudy as potential inhibitors of the protein/protein interaction betweenG-CSF at its receptor site. Another objective was to test the capacityof these drugs to facilitate dimerization of the G-CSF-R to reproducecentral and/or peripheral actions of G-CSF, i.e., to serve as G-CSFmimetic drugs.

Drugs.

Ten compounds were utilized for the present study, as shown in TABLE 1.

TABLE 1 Chemical Names of Compounds Tested.  12-(5-chloro-2-heptyloxy)phenyl)-   1-(1H-imidazol-1-yl)propan-2-ol  22-(5-chloro-2-(octyloxy)phenyl)-1-   (1H-imidazol-1-yl)propan-2-ol  32-(5-chloro-2-(nonyloxy)phenyl)-   1-(1H-imidazol-1-yl)propan-2-ol  41-(5-chloro-2-hydroxyphenyl)-2-(1H-   imidazol-1-yl)ethan-1-one  51-(5-chloro-2-hydroxyphenyl)-2-(1H-   imidazol-1-yl)ethan-1-one  64-(5-methoxy-1H-indo1-3-yl)-3,4-   dihydro-[1,3,5]-triazino-  [1,2a]benzimidazol-2-amine  7 3-[4-(2-amino-3,4-dihydro-  [1,3,5]triazino[1,2-a]benzimidazol-4-yl)-   1H-pyrazol-3-yl]phenol  84-[3-(4-methoxyphenyl)-1H-pyrazol-4-yl]-  3,4-dihydro[1,3,5]triazino[1,2-   a]benzimidazol-2-amine  94-(5-fluoro-1H-indo1-3-yl)-3,4- dihydro-[1,3,5]triazino[1,2-a]benzimidazol-2-amine 10 4-[3-(3-nitrophenyl)-1H-pyrazol-4-yl]-3,4-dihydro[1,3,5]triazino[1,2-a]- benzimidazol-2-amine

G-CSF Receptor Binding Parameters Assessed with [¹²⁵I]-G-CSF.

Binding of [¹²⁵I]-G-CSF was measured using previously described method(Kondo, S., et al. Eur. J. Haematol. 1991, 46, 223-30) with somemodifications (Pennington, A., et al. Alzheimer's Disease andParkinsonism 2013, 3, 121). [¹²⁵I]-G-CSF (specific activity 46.55TBq/mmol) was purchased from Perkin Elmer (Waltham, Mass.). Cells wereincubated in 180 μL of binding medium containing 0.2% BSA, 5 mM MgSO₄,and 50 mM Hepes, pH 7.2 at a concentration of 1.5×10⁷ cells/mL and theappropriate concentration of [¹²⁵I]-G-CSF. Incubations were carried outat room temperature with periodic shaking to ensure continuous mixing ofcells and radioactive ligand. The incubation time of 2 h was estimatedfrom preliminary experiments as being found sufficient to reachequilibrium. Nonspecific binding of [¹²⁵I]-G-CSF was measured byincubations in the presence of a 100-fold molar excess of unlabeledG-CSF. At the end of the incubation, bound and free radio ligands werediscriminated by using separating oil according to previously publishedmethod (Dower, S. K., et al. J. Exp. Med. 1985, 162, 501-15). Namely, 80μL aliquots were sampled from the incubation mixture, each aliquot waslayered on 300 μL of separating oil placed in 500 μL polyethylene tubesand centrifuged for 5 min at 6000 rpm. The separating oil consisted of1.5 parts dibutyl phthalate and 1 part bis(2-ethylhexyl)phthalate(Aldrich-Sigma, St. Louis, Mo.). Bound and free ligand activities werecounted after cutting tubes in two pieces and placing the tips and tops,respectively into separate scintillation vials. Counting was performedon Beckman Coulter LS6500 scintillation counter. Glacial acetic acid wasemployed to solubilize the pellet. The specific binding was determinedfrom the amount of bound [¹²⁵I]-G-CSF blocked by competition with excessunlabeled G-CSF. The parameters of saturation binding experimentsincluding dissociation constant (Kd), maximum binding capacity (Bmax)and binding cooperativity (h) were calculated with GraphPad Prism 5software (La Jolla, Calif.) by using nonlinear regression analysis.

Competition Studies.

Cells, prepared as described above, were incubated in 180 μL of bindingmedium containing 0.2% BSA, 5 mM MgSO₄ and 50 mM Hepes, pH 7.2 at aconcentration of 1.5×10⁷ cells/mL and the appropriate concentration of[¹²⁵I]-G-CSF. This was followed by addition of increasing concentrationsof each study compound (10 to 3000 nM). After 2 hrs of incubation, theamount of bound and free [¹²⁵I]-G-CSF was determined, as describedabove. From these data, the parameters of competition for the receptorwere calculated with GraphPad Prism 5 software (La Jolla, Calif.). Eachconcentration was analyzed in triplicate and experiments were repeatedthree times. These experiments were conducted in human monocytic andneuronal cell lines.

Signal Transduction Triggered by G-CSF (Measurement of PKCVlIII and Bcl2with Western Blot).

THP-1 cells or SH-SY5Y cells 3×10⁶ cells in 5 mL media in a 25 cm² flaskwere treated with 100 ng/mL G-CSF or three different concentrations ofthe test drug for 24 h. In addition, a set of flasks were co-incubatedwith the combination of G-CSF and 3 concentrations of test drug. Wholecell lysates (60 mg) were separated on 10% polyacrylamide gelelectrophoresis-SDS (PAGE-SDS). Proteins were electrophoreticallytransferred to nitrocellulose membranes, blocked with 5% nonfat milkprepared on Tris buffered saline containing 0.1% Tween 20, washed andincubated with a polyclonal antibody against either Bcl2 (CellSignaling, Danvers, Mass.) or PKCδVIII-specific polyclonal antibody(Jiang, K., et al. Biochemistry 2008, 47, 787-797). The house keepinggene GAPDH was used as an internal standard. Following incubation withanti-rabbit IgG-HRP, enhanced chemiluminesence (Pierce™, ThermoScientific, Walthahm, Mass.) was used for detection and the gels wereanalyzed using UN-SCAN-IT™ software (Silk Scientific, Inc., Orem, Utah).

Radial Arm Water Maze (RAWM).

To study the cognitive effects of G-CSF in mice that had undergone mildto moderate controlled cortical impact (CCI), a RAWM task will beemployed. RAWM is a hippocampal-dependent, spatial learning task thatdoes not rely on locomotor ability or swimming speed (Vorhees andWilliams, Nat. Protoc. 2006, 1, 848-858). Baseline RAWM is conducted inall mice before CCR and repeated at day 7 and day 14 post-CCI. A six-armradial arm maze was placed into a water tank of approximately 100-cmdiameter and 25-cm height; a 5-cm-diameter platform was used. Theplatform was submerged 0.5 cm below the water surface, and thetemperature of the water was kept at 268° C. Mice were placed in thestart arm at the beginning of every trial, and the platform was locatedin the goal arm. Every animal had an assigned platform/arm locationthroughout acquisition of learning, yet the starting zone was randomlychanged per trial. A spatial training protocol was followed. Mice weregiven two blocks of five trials, each. Based on prior experience thenumber of animals in each study was determined by establishing necessarygroup sizes to reach statistical significance for behavior andhistological analysis in each study (Song, S., et al. J. Neurosci. Res.2016, 94, 409-23; Song, S., et al., Restor. Neurol. Neurosci. 2016). Apower calculation based on number of errors was made in finding theplatform in the RAWM by untreated Tg mice and NT mice was made usingexperimental data. An n=8 for each group of mice (Tg and NT) had a 90%power to detect a difference between means of 1.12 errors with asignificance level (alpha) of 0.05 (two-tailed).

Surgery and Controlled Cortical Impact (CCI).

Animals will undergo TBI using a controlled cortical impactor(Pittsburgh Precision Instruments, Inc, USA) using methods we havepreviously described (Song, S., et al. J. Neurosci. Res. 2016, 94,409-23; Song, S., et al., Restor. Neurol. Neurosci. 2016). Briefly,animals will initially receive Buprenorphine (0.05 mg/kg, s.c.) at thetime of anesthesia induction (with gasesous anesthesia). Once deepanesthesia is achieved (by checking for pain reflexes), individualanimals are fixed in a stereotaxic frame (David Kopf Instruments,Tujunga, Calif., USA). After exposing the skull, craniectomy(approximately 2 mm), to accommodate the impactor tip) is performed overthe right frontoparietal cortex (−0.5 mm anteroposterior and +0.5 mmmediolateral to bregma). The pneumatically operated TBI device (with aconvex tip diameter=2 mm) impacts the brain at a velocity of 6.0 m/sreaching a depth of 0.5 mm, 1.0 mm or 2.0 mm for mild, moderate andsevere TBI respectively, below the dura mater layer and remains in thebrain for 150 ms. The impactor rod is angled 15° to the vertical tomaintain a perpendicular position in reference to the tangential planeof the brain curvature at the impact surface. A linear variabledisplacement transducer (Macrosensors, Pennsauken, N.J.), connected tothe impactor, measures velocity and duration to verify consistency. Bonewax is used to cover the craniectomized region and the skin incisionsutured thereafter. Sham injury surgeries consist of animals exposed toanesthesia, scalp incision, craniectomy, and suturing. A computeroperated thermal blanket pad and a rectal thermometer allowedmaintenance of body temperature within normal limits. All animals areclosely monitored until recovery from anesthesia and over the next 3consecutive days.

Immunohistology and Quantitative Image Analyses.

Mice were anesthetized with 150 mg/kg ketamine and 15 mg/kg xylazine andthen transcardially perfused with 0.9% saline, followed by 4%paraformaldehyde. Brains were stored in 4% paraformaldehyde and weretransferred to 25% sucrose solution in 4% paraformaldehyde until theysank to the bottom. Then, brains were slowly immersed in isopentane(cooled on dry ice), left in isopentane for 20 sec, removed, placed on asmall piece of aluminum foil resting on powdered dry ice for 1-2 min (toallow the isopentane evaporate), and finally wrapped in the foil andstored at −80° C. until sectioning. Brains slices were cut 30 mL thickon a cryostat (Leica) set to 225° C. Every sixth coronal section wastaken from the corpus striatum (caudate/putamen) spanning 1.2 mm in theanterior-posterior direction (from Bregma+1.32 mm to Bregma=0, whichcorresponds to the beginning of the lateral ventricles to the anteriorcommissure). Serial sections were also cut from the hippocampus,starting from Bregma −1.28 to Bregma −2.92. Every sixth section was keptfor immunostaining. Selective immunostaining of astrocytes and microgliawas performed with antibodies to GFAP and Iba-1, respectively. Iba-1 isprotein that is specifically expressed in macrophages/microglia and isupregulated during the activation of these cells. Brain sections werepreincubated in phosphate-buffered saline (PBS) containing 10% normalserum (goat or donkey; Vector Laboratories, Burlingame, Calif.) and 0.3%Triton X-100 (Sigma, St. Louis, Mo.) for 30 min. The sections were thentransferred to a solution containing primary antibodies in 1% normalserum and 0.3% Triton X-100/PBS and incubated overnight at 4° C.

Estimates of Hippocampal Neurogenesis.

Cells in hippocampus that are double-labeled with BrdU/nestin, BrdU/DCX,or BrdU/NeuN will be counted to determine extent of neurogenesis.Labeled cells will be visualized with fluorescence microscopy usingappropriate filters or with Zeiss LSM 510 confocal fluorescencemicroscope. Unbiased estimates of the number of doubly labeled BrdU+cells in dentate gyrus is made by counting in serially sectionedhippocampus according to the method previously described (Shors, T. J.,et al. Nature 2001, 410, 372-376; Shors T. J., et al. Hippocampus 2002,12, 578-584). Briefly, cell counts are estimated based on sectioning andcounting positively labeled cells in every 6th section (30 μm thick) oftissue (180 μm). A modification to the optical dissector method was usedso that cells on the upper and lower planes were not counted to avoidcounting partial cells. The number of BrdU+ cells counted in every 6thsection was multiplied by 6 to get the total number of BrdU+ cells inthe dentate gyrus. For the quantification of double labeled cells usingimmunofluroescence, the number of BrdU+ and BrdU+NeuN+ labeled cells isestimated using every 12th section taken throughout the dentate gyrus.Positive labeling is verified by confocal microscopy (Zeiss LSM510).Cells determined to be BrdU+ and BrdU+NeuN+ positive are tallied andmultiplied by the number of intervening sections.

The specific antibodies used will be rabbit anti-lba-1 (1:500; catalogNo. 019-19741; RRID:nlx_152487; Wako, Osaka, Japan), rabbit anti-GFAP(1:50 in PBS; catalog No. 60-0032-7; RRID:AB_11203520; Genemed, SanFrancisco, Calif.), and rabbit anti-DCX (1:1,000 containing 1:100 normalserum without Triton X-100; catalog No. 18723; RRID:nlx_152244; Abcam,Cambridge, Mass.). After incubation with primary antibody, sections arewashed and incubated for 1 hour with Alexa Fluor 488 goat anti-rabbitIgG diluted 1:400 in PBS (catalog No. A11070; RRID:AB_142134; MolecularProbes/Invitrogen, Carlsbad, Calif.) at room temperature. Sections werethen rinsed in PBS three times and covered with a coverglass. Greenfluorescence signals from the labeled cells are visualized byfluorescence microscopy with appropriate filters. For quantitative imageanalyses, all images will be acquired using an Olympus BX60 microscopewith an attached digital camera system (DP-70, Olympus, Tokyo, Japan),and the digital image will be routed into a Windows PC for quantitativeanalysis using ImageJ (NIH). To evaluate microglial burden (Ibalimmunoreactivity), after the mode of all images is converted to grayscale, the average intensity of positive signals from each image will bequantified in the CA1 and CA3 regions of hippocampus as a relativenumber from zero (white) to 255 (black). Each analysis is done by asingle examiner blinded to sample identities.

Intracellular Signaling Studies.

The experiments will be performed using methods previously publishedfrom the Patel lab (Apostolatos, H., et al. J. Biol. Chem. 2010, 285,25987-25995; Patel, N. A., et al. Gene Expression 2006, 13, 73-84).Briefly, the mouse splice variant PKCδII is generated by utilization ofan alternative downstream 5′ splice site of PKCδ pre-mRNA exon 9 (FIG.7). PKCδII is resistant to cleavage by caspase-3. PKCδII promotessurvival while PKCδI promotes apoptosis. PKCδVIII is the human homologof PKCδII, and both splice variants promote survival. For quantitativeimage analyses, all images will be acquired using an Olympus BX60microscope with an attached digital camera system (DP-70, Olympus,Tokyo, Japan), and the digital image will be routed into a Windows PCfor quantitative analysis using ImageJ (NIH). To evaluate microglialburden (Ibal immunoreactivity), after the mode of all images isconverted to gray scale, the average intensity of positive signals fromeach image will be quantified in the CA1 and CA3 regions of hippocampusas a relative number from zero (white) to 255 (black). Each analysis isdone by a single examiner blinded to sample identities.

Signal Transduction Data Analysis.

This study elucidates a novel aspect of G-CSF signaling in the brain.The role of Akt kinase in the cell is complex as it can activatemultiple cascades thereby regulating proteins involved in transcriptionand translation. G-CSF signaling is an intricate network of kinases andits substrates which change specificities depending upon the stimulus.In the event that Akt siRNA proves to be detrimental for the NSCs,experiments will be carried out using AKT1, AKT2, and AKT3 null mice.Alternatively, inducible RNAi systems will be used (Knockout SingleVector Inducible RNAi system, Clontech, Mountain View, Calif.) whichallow regulating the expression of functional short hairpin RNAs(shRNAs) in mammalian cells for the purpose of silencing target genes.The system is designed so that expression of an shRNA is induced wheneither tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is addedto the culture medium. Induction of the shRNA results in suppression ofthe gene targeted by the shRNA through RNAi. An Akt inhibitor (ML9) andClk/Sty inhibitor (TG003) may be used. To increase the transfectionefficiency in cells, an inducible adenovirus PKCδII construct may beused, such as PKCδII cloned into the VIRAPOWER™ Adenoviral ExpressionSystem (Invitrogen, Carlsbad, Calif.) which contains the pAd/CMV/V5-DESTGATEWAY®-adapted (Invitrogen, Carlsbad, Calif.) adenoviral vectorenabling high level protein expression from human cytomegalovirus (CMV)immediate-early enhancer/promoter.

Statistical Analysis.

Densitometric results from Western blots were analyzed with two-tailedt-tests when only two groups were compared. In other analyses where 3groups were compared, one-way ANOVA followed by t-tests with Dunnett'scorrections for multiple comparisons was utilized. All data waspresented as mean±SEM. All statistical analysis of data was done viaPRISM4 or PRISM5 statistical analysis software (GraphPad Prism, LaJolla, Calif.). Neurohistologic analyses are performed using ANOVA. Allcomparisons were considered significant at level of P<0.05.

Example 2 Computer in Silico Screening Identifies Potential G-CSFMimetics

Schödinger 3D-modeling software (https://www.schrodinger.com/smdd/) wasused to import a molecular model of G-CSF bound to its receptor from theNCBI protein database (PDB ID No. 1PGR_C;http://www.ncbi.nlm.nih.gov/protein/1PGR_C). The crystal structurecoordinates showed a point of interaction whereby a glutamate (Glu20)from G-CSF protrudes into a small pocket of G-CSF-R, which was estimatedto be a hydrogen bond accepting region. A molecule capable ofinterrupting this interaction could reduce binding of G-CSF withG-CSF-R. The first three compounds to be modeled in this system werethose originally discovered by Kusano (Blood 2004, 103, 836-42), whoreported their capacity to stimulate blood stem cell proliferation andto increase levels of circulating leukocytes in a rodent model. Thesethree compounds were observed to fit a narrow binding pocket nestledbetween G-CSF and the G-CSF-R. The three compounds were able to occupythe pocket with negative but high estimated free energies of binding.The occlusion of the binding site would impede the ability of G-CSF tointeract with G-CSF-R. The binding patterns indicated two points ofbinding that may be manipulated: two discrete binding sites at theprotein-protein interface and binding into a pocket to disrupt theinteraction of Glu20 from G-CSF with Tyr78 and Arg193 from G-CSF-R.Additional modeling identified 7 more compounds, 2 of which contained asimilar pharmacaphore as the original Kusano compound whereas the other5 were from an entirely different pharmacaphore. These compounds fit thebinding pocket between the natural G-CSF ligand and its receptor.Compounds 1-5 were synthesized by the Moffitt Cancer Center Chemistryunit, whereas compounds 6-10 were purchased from Chembridge Corporation(San Diego, Calif.).

Example 3 Characterization of the Binding of [¹²⁵I]-G-CSF to itsReceptor on Monocytes (THP-1 Cells) and Neuronal (SH-SY5Y) Cells

Increasing concentrations of radio-labeled G-CSF (alone or in thepresence of 100 fold excess “cold” G-CSF) were added to the cellcultures to generate saturation curves. Specific binding of [¹²⁵I]-G-CSFto receptors expressed in monocytes (THP-1 cells) revealed a Kd=57.49 pMand a Hill coefficient of 0.92. The Kd in the neuronal cell line(SH-SY5Y cells) was 607 pM with cooperative binding with a Hillcoefficient of 2.7 (see TABLE 2).

TABLE 2 Specific Binding Parameters of [¹²⁵I]-G-CSF to its receptor onTHP-1 cells and SH-SY5Y Cells. Monocytic Neuronal (THP-1) Cells(SH-SY5Y) Cells Bmax 901 pM 4826 pM Hill Coefficient 1.19 2.71 Kd 53.8pM 608 pM

The binding parameters, including dissociation constant (Kd), maximumbinding capacity (Bmax), and the Hill Coefficient were calculated withGraphPad Prism 5 software by using nonlinear regression analysis.

Binding of the ligand with G-CSF receptors expressed by monocytic cellsrevealed greater affinity (lower Kd) than binding to the neuronal cells.However, binding of the ligand receptors expressed by the neuronal lineexhibits substantial “cooperativity”, indicated by Hill coefficient of2.7 compared to 1.19 in the monocytic cell line. Cooperative bindingrefers to enhanced binding of ligand to a macromolecule when there arealready other ligands present on the same macromolecule. The Hillcoefficient describes the fraction of the receptor macromoleculesaturated by ligand as a function of the ligand concentration; it isused to determine the degree of cooperative binding of the ligand toreceptor. A coefficient of 1 indicates completely independent binding,regardless of how many additional ligands are already bound asdemonstrated in the saturation curve of the monocytic cell line. Numbersgreater than one indicate positive cooperativity, while numbers lessthan one indicate negative cooperativity.

Displacement studies were then conducted for each of the 10 compounds. Atypical displacement study result is shown in FIG. 2 demonstrating thecapacity of Compounds 6 and 10 to displace ¹²⁵I-G-CSF from its receptoron monocytic cells with an IC50 of 13.7 nM and 2.3 nM, respectively.Four of the ten compounds tested were capable of significantly competingwith G-CSF for binding on the receptor (FIG. 3A and FIG. 3B). Compounds6 and 10, which displaced greater than 75% of the ¹²⁵I-G-CSF, werefurther studied for their capacity to mimic or block the intracellularsignaling triggered by the natural ligand.

Binding of G-CSF to its receptor triggers receptor homo-dimerization andactivation of complex signal transduction and anti-apoptotic pathwayswhich increase expression of STAT3, Bcl-2, and decrease expression ofBax. Previous work in our laboratory had shown that G-CSF upregulatesexpression of PKCδVIII and Bcl2 in human monocytic and neuronal cellcultures. G-CSF was added to the cell cultures and incubated for 24hours. Whole cell lysates were immunoblotted for Bcl2 and PKCδVIII.Despite the much lower binding affinity of G-CSF for receptors expressedby neurons compared to binding affinity in monocytic cells, theexpression of the anti-apoptotic protein Bcl2 and PKCδVIII, was muchgreater at equivalent concentrations (100 ng/mL or 5.3 nM) in the humanneuronal cell line (FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D). G-CSF,however, also increased the expression of PKCδVIII and Bcl2 in themonocytic line at a higher concentration (200 ng/mL or 10.6 nM).

The two most effective competitors for the G-CSF receptor, Compounds 6and 10 were evaluated for their capacity to activate or block theintracellular signaling triggered by G-CSF in human neuronal cells(SH-SY5Y) (FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D). Incubation of humanneuronal cells with Compound 6 alone significantly increased Bcl2protein expression at the low and intermediate, but not the highconcentration (FIG. 5C). In addition, Compound 6 alone stimulatedPKCδVIII protein expression to levels equivalent to G-CSF alone.Incubation of the neuronal cells with both Compound 6 and G-CSF did notchange the expression of either Bcl2 or PKCδVIII, indicating thatCompound 6 did not act as an antagonist of G-CSF action in the neuronalcell line (FIG. 5C).

Incubation of the neuronal cell line with Compound 10 alone increasedexpression of both PKCδVIII and Bcl2 protein to levels equivalent toG-CSF alone (FIG. 5D). In addition, Compound 10 at all threeconcentrations tested did not block the actions of G-CSF. To summarizeboth Compound 6 and Compound 10 appear to act as agonists at the G-CSFreceptor while Compound 6 at the highest concentration increasesPKCδVIII without changing Bcl2 expression.

Studies in the monocytic THP-1 cell line revealed a different profile ofdrug-induced receptor activation (FIG. 6A, FIG. 6B, FIG. 6C, and FIG.6D). Compound 6 alone increased expression of Bcl2, but not PKCδVIII atall 3 concentrations. Unlike the results in the neuronal cell line,addition of Compound 6 to G-CSF blocked the increase in Bcl2 andPKCδVIII protein expression (FIG. 6C). Hence, Compound 6 appears to actas a mixed agonist/antagonist in monocytic cell lines. Compound 10, atall 3 concentrations, was effective in significantly elevatingexpression of both Bcl2 and PKCδVIII (FIG. 6D). Compound 10 alsoappeared to act as a mixed agonist/antagonist by blocking the effects ofG-CSF on Bcl2 expression. However, Compound 10 did not antagonize theeffects on PKCδVIII protein expression.

Based on computer-based modeling of Compounds 1-3 and a search of druglibraries, we identified two pharmacophores with five analogues each fora total of ten compounds with potential to interact with the G-CSFreceptor in human cell culture lines. Two of the ten drugs (Compound 6and Compound 10) purchased from a commercial supplier were shown tointeract with the G-CSF receptor and to trigger signal transduction inhuman monocytic and neuronal human cell cultures. Compound 10 appearedto function as a G-CSF agonist in neuronal cell lines. Both Bcl2 andPKCδVIII protein expression was increased by treatment with the drug inthe neuronal cells. By contrast, Compound 10 at the lowest concentrationantagonized the effects of G-CSF on the expression of Bcl2 and PKCδVIIIin the monocytic cell lines. Therefore it may be possible to develop acombination drug (for example, G-CSF+Compound 10) that will serve as aneurotrophic factor in vivo, free of potentially dangerous leukocytosis.This could be accomplished by stimulating neurons that bear G-CSFreceptor (for example, by both Compound 10 and G-CSF). At the same timeCompound 10 would be capable of blocking the peripheral effects of G-CSFand thereby prevent excessive leukocytosis.

We have found compounds that mimic the neurotrophic and/orimmune-modulating actions of G-CSF. The compounds are easier and cheaperto make than human G-CSF. Selective stimulation of central nervoussystem G-CSF receptors may be beneficial for treating some CNS diseasesin which excessive generation of leukocytes and thrombocytes is anunwanted side effect. In addition, G-CSF receptor antagonistsimpermeable to brain may be useful in distinguishing peripheral fromcentral actions of G-CSF. G-CSF antagonists may also be therapeutic forblood diseases associated with over-activity of the G-CSF system.

Example 4 Assessing the Capacity of Compounds to Mimic or Block theIntracellular Signaling Triggered by G-CSF

Signal Transduction Induced by Stimulation of the G-CSF Receptor.

THP-1 cells or SH-SY5Y cells 3×10⁶ cells in 5 mL media in a 25 cm² flaskwill be treated with 100 ng/mL G-CSF for 24 hours in the presence ofincreasing concentrations of the compounds to be tested. In parallelcultures, the compounds will be added to the cultures without G-CSF for24 hours. Whole cell lysates (60 mg) will be separated on 10%polyacrylamide gel electrophoresis-SDS (PAGE-SDS). Proteins will beelectrophoretically transferred to nitrocellulose membranes, blockedwith Tris buffered saline, 0.1% Tween 20 containing 5% nonfat driedmilk, washed, and incubated with a polyclonal antibody against eitherBcl2 (Cell Signaling, Danvers, Mass.) or PKCδVIII (Chen, Y. K., et al.J. Vasc. Surg. 2008, 47, 1058-65). The house keeping gene GAPDH will beused as an internal standard. Following incubation with anti-rabbitIgG-HRP, enhanced chemiluminesence (Pierce™, Thermo Scientific, Waltham,Mass.) will be used for detection, and the gels will be analyzed usingUN-SCAN-IT™ software (Silk Scientific, Inc., Orem, Utah).

Assessing the Expression of PKCδII Splice Variants Regulated by G-CSF.

The effect of G-CSF in the presence of the test compounds or the effectsof the test compounds alone on the expression of PKCδ isozymes in NSCwill be studied. We will evaluate a dose curve to determine the optimalconcentration. After 24 hours of incubation, total RNA will beextracted, and RT-PCR analysis will be performed with primers thatsimultaneously amplify PKCδI and PKCδII. Simultaneously, whole celllysates will be harvested, and western blot analysis will be performed.Previous results indicate PKCδII increased Bcl2 and phosphorylation ofBAD. We will evaluate the effect of G-CSF and the test compounds both atthe RNA and protein level to decipher whether it is a transcriptional ortranslational effect. GAPDH levels will serve as an internal control.

In separate experiments, we will silence PKCδII (Ambion pre-designedsiRNA, specificity determined in previous Patel publications) and thenevaluate the test compounds. This will enable us to determine ifadditional pathways are activated to compensate for the loss of PKCδII.RT-qPCR and western blot analysis will be performed. We will alsoimmunoblot for additional apoptosis indicators such as PARP and XIAP.

Further Probing of Signal Transduction Pathways Induced by G-CSFReceptor Activation.

To determine which signal transduction pathways are involved inG-CSF-mediated PKC (alternative splicing in neuronal cells, inhibitorsof several signal transduction pathways will be applied. Inhibitors willbe added 30 minutes prior to the addition of G-CSF or the testcompounds. Signal transduction inhibitors to be used include thefollowing: (a) LY29402 (a PI3K inhibitor); (b) Rapamycin (p70/85 S6kinase inhibitor); (c) Herbimycin (a tyrosine kinase inhibitor); and (d)Ruxolitinib (JAK/STAT inhibitor). We will also determine thephosphorylation state of Akt kinase because it is a key downstreammediator of the PI3K pathway.

Once we determine the pathway involved, we will use pre-validatedspecific siRNAs for PI3K or JAK and STAT. 10 to 50 nM of siRNA will betransiently transfected for 48 to 72 hours into NSC and RT-PCR performedas described above. Scrambled siRNA will be used as control foroff-target silencing. As such, the data will be evaluated using twoseparate approaches: one molecular and the other pharmacological.

In experiments in which LY294002 is added prior to incubation withG-CSF, we expect a decrease in phosphorylation of Akt kinase (Ser 473)with concurrent decreased PKCδII levels. We expect total Akt levels willremain the same, indicating that G-CSF mediated its effect via thePI3K→Akt pathway. Test compounds that reproduce the same signalingcascade will be considered to be agonists (or least partial agonists) ofthe G-CSF receptor. It is possible that a compound may have profoundeffects on PKCδII but lower effect on Bcl2. This may be due to the factthat additional pathways also activate Bcl2 cascade.

Example 5 In Vivo Expression of PKCδII in Mouse Brain Tissue from Micethat Undergo Controlled Cortical Impact (CCI)

Since G-CSF may increase neurogenesis via PKCδII (the mouse homolog ofhuman PKCδVIII), we will determine the expression levels of PKCδII inmice treated with or without G-CSF. One cohort (n=6) of mice willreceive G-CSF injections subcutaneously every other day for 2 weeks, andthe other group will receive vehicle control (saline). In parallelexperiments, 3 different concentrations of a test compound will beinjected in mice daily for 1 week (6 mice per dose; total of 24 mice forthe test compound). Brain tissue will be harvested, hippocampusdissected, followed by total RNA isolation for RT-PCR analysis. Theprimers will amplify PKCδI and PKCδII levels simultaneously. Westernblot analysis will be performed on whole cell lysates as describedabove. We expect G-CSF will increase PKCδII expression levels in vivo,while PKCδI expression will remain unaffected. A compound as detailedherein with capacity to induce PKCδII in the NSC cultures should producethe same result.

Potential pitfalls and alternative approaches: The small molecule mayfail to trigger signal transduction in brain tissue simply because itfails to cross the blood-brain-barrier. This can be tested bystereotaxic micro-injection of the drug into hippocampus or other brainregion. If indeed direct injection of the test compound into brainreproduces the signal transduction effects of C-CSF administration, thenthe effects of the drug may be evident in the periphery. This can beassessed by measuring numbers of circulating hematopoietic stem cells(SCA-1+, c-kit+ cells), which may be significantly increased if itmimics the actions of G-CSF.

Example 6 Effects on Promotion of Recovery in a Hippocampal-DependentTask (RAWM) and on the Increased Expression of Neurotrophic Factors(BDNF and GDNF) in a Mouse Model of TBI

G-CSF treatment significantly promoted recovery in the radial arm watermaze (RAWM). We will test the capacity of compounds detailed herein toreproduce this effect. A cohort of C57BL6J mice will be trained in theRAWM. Then mice will undergo controlled cortical impact (CCI) describedin Example 1. Mice will then be randomly assigned to 8 cohorts. Eachcohort will be injected i.p. daily for 3 days with a test compound at 3doses or vehicle and in combination with G-CSF (n=8 per dose; total 64mice). See TABLE 3 for cohorts to be treated with Compound 6. Mice willbe re-tested in the RAWM at one and two weeks after CCI beforeundergoing euthanasia. Before intracardiac perfusion, blood samples willbe collected (to measure extent of leukocytosis and monocytosis). Thebrain will be perfused with PBS and phosphate-buffered paraformaldehyde,followed by dissection into right and left cortex, striatum, andhippocampus. These brain regions will be processed for measurements oftwo neurotrophic factors (BDNF, GDNF) and signal transduction assays(Bcl2 and PKCδII). Signal transduction and neurotrophic factor assaysare described in Example 1, as well as the FACS protocol for countingblood leukocytes and monocytes. The same experimental protocol will berepeated with Compound 10.

TABLE 3 Effects on RAWM and Bcl2/PKCδIII expression. Cohorts TreatmentFrequency N A Vehicle alone 1 per day × 3  8 B G-CSF (100 μg/kg) +saline 1 per day × 3  8 C Compound 6 (1 mg/kg) 1 per day × 3  8 DCompound 6 (10 mg/kg) 1 per day × 3  8 E Compound 6 (30 mg/kg) 1 per day× 3  8 F G-CSF 100 μg/kg + Compound 6 (1 mg/kg) 1 per day × 3  8 G G-CSF100 μg/kg + Compound 6 (10 mg/kg) 1 per day × 3  8 H G-CSF 100 μg/kg +Compound 6 (30 mg/kg) 1 per day × 3  8 Total n = 64

The extent to which the test compound improves RAWM performance andactivates G-CSF receptor-mediated signal transduction and increasesexpression of neurotrophic factors depends on how similar the biologicalactivity of the drug mimics the natural ligand G-CSF, including itsability to access the CNS. Based on our cell culture data, Compound 10administered alone will likely stimulate Bcl2 expression in both neuralcells of brain and peripheral blood cells and hence one would see a verysimilar response as G-CSF alone. However when Compounds 10 is given withG-CSF, only the central nervous system effects will be observed. Henceit is theoretically possible to effect a direct central nervous systembenefit by use of G-CSF plus Compound 10. In addition, Compound 6 willbe studied in combination with G-CSF. We expect administration ofCompound 6 will result in antagonism of G-CSF on peripheral cells whileat the same time stimulate brain G-CSF receptors.

Failure of the drug to improve performance in the RAWM might reflectlimited penetration of the drug into the CNS. An alternative approachwould be to micro-inject the drug directly into hippocampus or striatum.If direct injection of the test compound into brain reproduces theeffect of systemic G-CSF administration on signal transduction andneurotrophic factor expression, then, it may indicate that directactions of G-CSF in brain (especially hippocampus) are necessary toenhance recovery in the RAWM. In other words, it may suggest that theactions of G-CSF on its receptor in the periphery (bone marrow cells)may not be paramount for mediating beneficial effects in brain.Pharmacokinetic studies to determine plasma half-life and extent ofpenetration into CNS may be done.

Example 7 Effects on Hippocampal Neurogenesis and Microgliosis In VivoFollowing TBI

Following controlled cortical impact (CCI) on day 0, Mice C57BL6J willbe randomly assigned to 8 cohorts. All mice will be injected with 75mg/kg bromodeoxyuridine (BrdU) i.p. daily x 3 days along with the testdrug (with and without G-CSF as shown in TABLE 4 for cohorts to betreated with Compound 6).

Two weeks after the last dose of G-CSF or drug (enough time for newlyborn cells to differentiate into neurons), mice will be euthanized,animals perfused, and the brain dissected into right and left frontalcortex, striatum, and hippocampus. Tissue is then prepared forcryosectioning and immunohistology. Methods for immunostaining,stereologic estimates of neurogenesis and measurement of microgliosisare described in Example 1. The same experimental protocol will berepeated with Compound 10.

TABLE 4 Effects on Neurogenesis and Microgliosis. Cohorts TreatmentFrequency N A Vehicle alone 1 per day × 3  8 B G-CSF (100 μg/kg) +saline 1 per day × 3  8 C Compound 6 (1 mg/kg) 1 per day × 3  8 DCompound 6 (10 mg/kg) 1 per day × 3  8 E Compound 6 (30 mg/kg) 1 per day× 3  8 F G-CSF 100 μg/kg + Compound 6 (1 mg/kg) 1 per day × 3  8 G G-CSF100 μg/kg + Compound 6 (10 mg/kg) 1 per day × 3  8 H G-CSF 100 μg/kg +Compound 6 (30 mg/kg) 1 per day × 3  8 Total n = 64

The total number of BrdU+ nuclei will be significantly increased in micetreated with (1) G-CSF or (2) Compound 6 alone, or (3) Compound 10alone, indicating that the G-CSF or its mimetics stimulate neuralstem/progenitor cell proliferation. Co-administration of Compound 6 withG-CSF will result in antagonism of G-CSF on peripheral cells while atthe same time stimulating brain G-CSF receptors. By counting the totalnumber of doubly-labeled BrdU+/NeuN (neuron-specific nuclear protein)nuclei, G-CSF treatment or G-CSF mimetics will significantly increasenumber of new neurons in hippocampus compared to vehicle-treated mice.

The test compound may fail to stimulate neurogenesis simply because itfails to cross the blood-brain-barrier. This can be tested bystereotaxic micro-injection of the drug into hippocampus. If indeeddirect injection of the test compound into brain reproduces theneurogenic effects of C-CSF administration, then the structure of thecompound may be modified to make it permeable to brain.

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent disclosure. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A method of treating a condition in a subject, the methodcomprising administering a compound to the subject, wherein the compoundis according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula II:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy, and wherein thecondition is selected from the group consisting of neurodegenerativedisease, stroke, traumatic brain injury (TBI), impaired motor function,and impaired cognitive function.

Clause 2. The method of clause 1, wherein the neurodegenerative diseaseis selected from the group consisting of Alzheimer's Disease (AD),amyotrophic lateral sclerosis (ALS), Parkinson's Disease (PD), priondisease, motor neuron disease, Huntington's Disease, spinocerebellarataxia, and spinal muscular atrophy.

Clause 3. A method of stimulating the central nervous system GranulocyteColony-Stimulating Factor (G-CSF) Receptor in a subject, the methodcomprising administering a compound to the subject, wherein the compoundis according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula II:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,

or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy.

Clause 4. The method of any one of clauses 1-3, wherein the compound isof Formula I, and wherein R¹ and R² are each hydrogen.

Clause 5. The method of any one of clauses 1-3, wherein the compound isof Formula I, and wherein R¹ and R² together with the carbon atoms towhich they are attached form a phenyl ring.

Clause 6. The method of any one of clauses 1-3, wherein the compound isof Formula I, and wherein R³ and R⁴ are each hydrogen.

Clause 7. The method of any one of clauses 1-3, wherein the compound isof Formula I, and wherein R³ and R⁴ together with the atoms to whichthey are attached form a six-membered heterocyclic ring, wherein thering is unsubstituted or substituted with one substituent selected fromamino, nitro, methyl, ethyl, and hydroxyl.

Clause 8. The method of clause 7, wherein the six-membered heterocyclicring is substituted with amino or nitro.

Clause 9. The method of any one of clauses 1-3, wherein the compound isof Formula I, and wherein X is —C(R⁵)(R⁶)-phenyl wherein the phenyl issubstituted with R⁷ and R⁸.

Clause 10. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula III, and wherein R⁵ and R⁶ are eachindependently hydroxyl or methyl.

Clause 11. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula III, and wherein R⁵ and R⁶ together form an oxogroup.

Clause 12. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula III, and wherein R⁷ is hydroxyl or C₁-C₁₂alkoxy.

Clause 13. The method of clause 12, wherein R⁷ is hydroxyl.

Clause 14. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula III, and wherein R⁸ is halogen.

Clause 15. The method of clause 14, wherein halogen is Cl.

Clause 16. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula II, and wherein X is indole substituted with 0,1, 2, or 3 R⁹.

Clause 17. The method of clause 16, wherein each R⁹ is independentlyhalogen or methoxy.

Clause 18. The method of any one of clauses 1-3, wherein the compound isof Formula I or Formula II, and wherein X is pyrazole substituted withphenyl, wherein the phenyl is substituted with 0, 1, 2, or 3 R¹⁰.

Clause 19. The method of clause 18, wherein each R¹⁰ is independentlyhydroxyl, methoxy, or nitro.

Clause 20. The method of any one of the previous clauses, wherein thecompound decreases amyloid burden, enhances neurogenesis, enhancessynaptogenesis, or enhances cognitive performance, or a combinationthereof.

Clause 21. The method of any one of the previous clauses, wherein thecompound binds the Granulocyte Colony-Stimulating Factor (G-CSF)Receptor.

Clause 22. The method of any one of the previous clauses, wherein thecompound displaces at least 50% of G-CSF from the G-CSF receptor.

Clause 23. The method of clause 6, wherein the compound displaces atleast 75% of G-CSF from the G-CSF receptor.

Clause 24. The method of any one of the previous clauses, wherein thecompound is a peripheral antagonist of the G-CSF receptor.

Clause 25. The method of any one of the previous clauses, wherein thecompound is a central agonist of G-CSF receptor.

Clause 26. The method of any one of the previous clauses, whereinexpression of Bcl2 is increased.

Clause 27. The method of any one of the previous clauses, whereinexpression of PKCδVIII is increased.

Clause 28. The method of any one of the previous clauses, whereinexpression of STAT3 is increased.

Clause 29. The method of any one of the previous clauses, whereinexpression of Bax is decreased.

Clause 30. The method of any one of the previous clauses, whereinleukopoiesis is minimally affected or is not affected.

Clause 31. The method of any one of clauses 1-3 and 20-30, wherein thecompound is selected from the following:

Clause 32. The method of any one of clauses 1-3 and 20-30, wherein thecompound is selected from the following:

Clause 33. The method of any one of clauses 1-3 and 20-30, wherein thecompound is selected from the following:

Clause 34. The method of any one of the above clauses, wherein thecompound is co-administered with a G-CSF polypeptide.

Clause 35. The method of clause 34, wherein the G-CSF polypeptidecomprises an amino acid sequence of SEQ ID NO: 1.

Clause 36. The method of clause 34, wherein the G-CSF polypeptide isencoded by a polynucleotide of SEQ ID NO: 2

Clause 37. A compound selected from the following:

SEQUENCES Polypeptide sequence of human G-CSF. Accession No. CAA27291SEQ ID NO: 1    1MAGPATQSPM KLMALQLLLW HSALWTVQEA TPLGPASSLP QSFLLKCLEQ VRKIQGDGAA   61LQEKLVSECA TYKLCHPEEL VLLGHSLGIP WAPLSSCPSQ ALQLAGCLSQ LHSGLFLYQG  121LLQALEGISP ELGPTLDTLQ LDVADFATTI WQQMEELGMA PALQPTQGAM PAFASAFQRR  181AGGVLVASHL QSFLEVSYRV LRHLAQPPolynucleotide sequence of human G-CSF (mRNA). Accession No. E01219SEQ ID NO: 2    1ggagcctgca gcccagcccc acccagaccc atggctggac ctgccaccca gagccccatg   61aagctgatgg ccctgcagct gctgctgtgg cacagtgcac tctggacagt gcaggaagcc  121acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg cttagagcaa  181gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctgtgtgc cacctacaag  241ctgtgccacc ccgaggagct ggtgctgctc ggacactctc tgggcatccc ctgggctccc  301ctgagcagct gccccagcca ggccctgcag ctggcaggct gcttgagcca actccatagc  361ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc cgagttgggt  421cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat ctggcagcag  481atggaagaac tgggaatggc ccctgccctg cagcccaccc agggtgccat gccggccttc  541gcctctgctt tccagcgccg ggcaggaggg gtcctagttg cctcccatct gcagagcttc  601ctggaggtgt cgtaccgcgt tctacgccac cttgcccagc cctgagccaa gccctcccca  661tcccatgtat ttatctctat ttaatattta tgtctattta agcctcatat ttaaagacag  721ggaagagcag aacggagccc caggcctctg tgtccttccc tgcatttctg agtttcattc  781tcctgcctgt agcagtgaga aaaagctcct gtcctcccat cccctggact gggaggtaga  841taggtaaata ccaagtattt attactatga ctgctcccca gccctggctc tgcaatgggc  901actgggatga gccgctgtga gcccctggtc ctgagggtcc ccacctggga cccttgagag  961tatcaggtct cccacgtggg agacaagaaa tccctgttta atatttaaac agcagtgttc 1021cccatctggg tccttgcacc cctcactctg gcctcagccg actgcacagc ggcccctgca 1081tccccttggc tgtgaggccc ctggacaagc agaggtggcc agagctggga ggcatggccc 1141tggggtccca cgaatttgct ggggaatctc gtttttcttc ttaagacttt tgggacatgg 1201tttgactccc gaacatcacc gacgcgtctc ctgtttttct gggtggcctc gggacacctg 1261ccctgccccc acgagggtca ggactgtgac tctttttagg gccaggcagg tgcctggaca 1321tttgccttgc tggacgggga ctggggatgt gggagggagc agacaggagg aatcatgtca 1381ggcctgtgtg tgaaaggaag ctccactgtc accctccacc tcttcacccc ccactcacca 1441gtgtcccctc cactgtcaca ttctaactga acttcaggat aataaagtgc ttgcctccaa 1501aaaaaaaaaa aaaaaaaaaa a Polypeptide sequence of human G-CSF-RAccession No. Q99062 SEQ ID NO: 3    1MARLGNCSLT WAALIILLLP GSLEECGHIS VSAPIVHLGD PITASCIIKQ NCSHLDPEPQ   61ILWRLGAELQ PGGRQQRLSD GTQESIITLP HLNHTQAFLS CCLNWGNSLQ ILDQVELRAG  121YPPAIPHNLS CLMNLTTSSL ICQWEPGPET HLPTSFTLKS FKSRGNCQTQ GDSILDCVPK  181DGQSHCCIPR KHLLLYQNMG IWVQAENALG TSMSPQLCLD PMDVVKLEPP MLRTMDPSPE  241AAPPQAGCLQ LCWEPWQPGL HINQKCELRH KPQRGEASWA LVGPLPLEAL QYELCGLLPA  301TAYTLQIRCI RWPLPGHWSD WSPSLELRTT ERAPTVRLDT WWRQRQLDPR TVQLFWKPVP  361LEEDSGRIQG YVVSWRPSGQ AGAILPLCNT TELSCTFHLP SEAQEVALVA YNSAGTSRPT  421PVVFSESRGP ALTRLHAMAR DPHSLWVGWE PPNPWPQGYV IEWGLGPPSA SNSNKTWRME  481QNGRATGFLL KENIRPFQLY EIIVTPLYQD TMGPSQHVYA YSQEMAPSHA PELHLKHIGK  541TWAQLEWVPE PPELGKSPLT HYTIFWTNAQ NQSFSAILNA SSRGFVLHGL EPASLYHIHL  601MAASQAGATN STVLTLMTLT PEGSELHIIL GLFGLLLLLT CLCGTAWLCC SPNRKNPLWP  661SVPDPAHSSL GSWVPTIMEE DAFQLPGLGT PPITKLTVLE EDEKKPVPWE SHNSSETCGL  721PTLVQTYVLQ GDPRAVSTQP QSQSGTSDQV LYGQLLGSPT SPGPGHYLRC DSTQPLLAGL  781TPSPKSYENL WFQASPLGTL VTPAPSQEDD CVFGPLLNFP LLQGIRVHGM EALGSF

1. A method of treating a condition in a subject, the method comprisingadministering a compound to the subject, wherein the compound isaccording to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro, or the compound is according to FormulaII:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy, and wherein thecondition is selected from the group consisting of neurodegenerativedisease, stroke, traumatic brain injury (TBI), impaired motor function,and impaired cognitive function.
 2. The method of claim 1, wherein theneurodegenerative disease is selected from the group consisting ofAlzheimer's Disease (AD), amyotrophic lateral sclerosis (ALS),Parkinson's Disease (PD), prion disease, motor neuron disease,Huntington's Disease, spinocerebellar ataxia, and spinal muscularatrophy.
 3. A method of stimulating the central nervous systemGranulocyte Colony-Stimulating Factor (G-CSF) Receptor in a subject, themethod comprising administering a compound to the subject, wherein thecompound is according to Formula I:

wherein R¹ and R² are each hydrogen, or R¹ and R² together with thecarbon atoms to which they are attached form a phenyl ring; R³ and R⁴are each hydrogen, or R³ and R⁴ together with the atoms to which theyare attached form a six-membered heterocyclic ring, wherein the ring isunsubstituted or substituted with one substituent selected from amino,nitro, methyl, ethyl, and hydroxyl; X is —C(R⁵)(R⁶)-phenyl wherein thephenyl is substituted with R⁷ and R⁸, or X is indole substituted with 0,1, 2, or 3 R⁹, or X is pyrazole substituted with phenyl wherein thephenyl is substituted with 0, 1, 2, or 3 R¹⁰; R⁵ and R⁶ are eachindependently selected from hydrogen, hydroxyl, C₁-C₄ alkyl, and C₁-C₄alkoxy, or R⁵ and R⁶ together form an oxo group; R⁷ is hydrogen,hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; R⁸ is halogen, hydrogen,hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy; each R⁹ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro;and each R¹⁰ is independently halogen, hydrogen, hydroxyl, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, or nitro, or the compound is according to FormulaII:

wherein X is indole substituted with 0, 1, 2, or 3 R⁹, or X is pyrazolesubstituted with phenyl wherein the phenyl is substituted with 0, 1, 2,or 3 R¹⁰; each R⁹ is independently halogen, hydrogen, hydroxyl, C₁-C₄alkyl, C₁-C₄ alkoxy, amino, or nitro; and each R¹⁰ is independentlyhalogen, hydrogen, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, amino, or nitro,or the compound is according to Formula III:

wherein R⁵ and R⁶ are independently hydrogen, hydroxyl, methyl, ethyl,methoxy, or ethoxy, or R⁵ and R⁶ together form an oxo group; R⁷ ishydrogen, hydroxyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; and R⁸ is halogen,hydrogen, hydroxyl, C₁-C₄ alkyl, or C₁-C₄ alkoxy. 4-11. (canceled) 12.The method of claim 1, wherein the compound is of Formula I or FormulaIII, and wherein R⁷ is hydroxyl or C₁-C₁₂ alkoxy. 13-15. (canceled) 16.The method of claim 1, wherein the compound is of Formula I or FormulaII, and wherein X is indole substituted with 0, 1, 2, or 3 R⁹. 17.(canceled)
 18. The method of claim 1, wherein the compound is of FormulaI or Formula II, and wherein X is pyrazole substituted with phenyl,wherein the phenyl is substituted with 0, 1, 2, or 3 R¹⁰.
 19. (canceled)20. The method of claim 1, wherein the compound decreases amyloidburden, enhances neurogenesis, enhances synaptogenesis, or enhancescognitive performance, or a combination thereof.
 21. The method of claim1, wherein the compound binds the Granulocyte Colony-Stimulating Factor(G-CSF) Receptor.
 22. The method of claim 1, wherein the compounddisplaces at least 50% of G-CSF from the G-CSF receptor.
 23. (canceled)24. The method of claim 1, wherein the compound is a peripheralantagonist of the G-CSF receptor.
 25. The method of claim 1, wherein thecompound is a central agonist of G-CSF receptor.
 26. The method of claim1, wherein expression of Bcl2 is increased.
 27. The method of claim 1,wherein expression of PKCδVIII is increased.
 28. (canceled)
 29. Themethod of claim 1, wherein expression of Bax is decreased.
 30. Themethod of claim 1, wherein leukopoiesis is minimally affected or is notaffected.
 31. The method of claim 1, wherein the compound is selectedfrom the following:


32. The method of claim 1, wherein the compound is selected from thefollowing:


33. The method of claim 1, wherein the compound is selected from thefollowing:


34. The method of claim 1, wherein the compound is co-administered witha G-CSF polypeptide. 35-36. (canceled)
 37. A compound selected from thefollowing: